Isolation of adult multipotential cells by tissue non-specific alkaline phosphatase

ABSTRACT

The present invention relates to the use of tissue non-specific alkaline phosphatase (TNAP) as a marker for identifying and/or isolating adult multipotential cells. The present invention also relates to cell populations enriched by methods of the present invention and therapeutic uses of these cells.

FIELD OF THE INVENTION

The present invention relates to the use of tissue non-specific alkalinephosphatase (

TNAP) as a marker for identifying and/or isolating adult multipotentialcells. The present invention also relates to cell populations enrichedby methods of the present invention and therapeutic uses of these cells.

BACKGROUND OF THE INVENTION Enrichment of Adult Multipotential Cells

Numerous studies support the concept that the non-haemopoietic cells ofthe bone marrow (BM), which include fibroblasts, adipocytes,choiadroblasts, smooth muscle cells, osteoblasts and other cellularelements of bone, are derived from a population of multipotential bonemarrow mesenchymal precursor cells (MPC), residing somewhere in the bonemarrow spaces and the surrounding connective tissue (Bianco et al.,2001; Gronthos and Simmons, 1996; Owen and Friedenstein, 1988; Prockop,1997). These MPC are thought to give rise not only to more cells whichare phenotypically and functionally identical (a process ofself-renewal), but also differentiated, lineage-committed mesenchymalprogeny. Due to the lack of well defined markers, little is known of theprecise developmentally regulated changes in phenotype and patterns ofgene expression, which occur during the differentiation and maturationof human MPC into lineage-committed progeny. Studies examining theprocess of osteogenesis have identified one such early marker, thetranscription factor CBFA1, which enables the identification of MPCwhich have made a commitment to the osteogenic cell lineage (Ducy et al,1997). However, markers such as CBFAI, can not be used to isolate andmanipulate living cells within a heterogeneous cell population. Thisrepresents a major limitation, and is further compounded by a paucity ofmonoclonal antibodies (mAb) which are able to identify cell surfaceantigens which are peculiar to or restricted to the MPC compartment.

To date, the STRO-1 monoclonal antibody represents the only reagentwhich demonstrates immunoreactivity with all colony forming MPC (CFU-F:colony-forming units-fibroblasts) from aspirates of human marrow whilstlacking reactivity with haemopoietic stein cells (Dennis et al., 2002;GTonihos et al., 2003; Simmons and Torok-Storb, 1991).

Our studies have shown that er viva expanded human MPC quicklydifferentiate in the presence of serum, and begin expressing many of themarkers associated with commitment to the osteogenic and other celllineages (Gronthos et al., 2003). The mAb STRO-1 which identifies allMPC (CFU-F) in vivo, is down regulated following ex vivo culture of MPC.Importantly, a small proportion of cultured cells continue to expressSTRO-1 following ex vivo expansion and these cells are characteristic ofundifferentiated MPC (Gronthos et al., 1999; Stewart et al., 1999).

Alkaline Phosphatases

Alkaline phosphatases (AP, EC 3.1.3,1) belong to a ubiquitous family ofdimeric metalloenzymes which catalyse the hydrolysis ofphosphomonoesters under alkaline conditions with release of inorganicphosphate (McComb et al., 1979). One can distinguish between fourisoenzymes in humans: i) placenta-specific AP, ii) germ cell specific(placental) AP, iii) intestinal AP and iv) the tissue non-specific AP(TNAP) (Harris, 1990). The production of TNAP is strongest in the liver(LAP), kidney (KAP) and bones (BAP) (Moss, 1992) and is the mostfrequent AP isoform in serum (Mulivor, et al., 1985). The differencesbetween LAP, KAP and BAP are due to different posttranslationalO-glycosylation patterns (Miura, et al., 1994) which also results indifferent specific activities (Nosjean et al., 1997) although theiramino acid sequences are essentially identical (Weiss et al., 1988).Furthermore Nosjean et al. (1997) have shown that the N-glycosylation oftns-AP is essential for its enzymatic activity. Consequently tissuenon-specific AP is a mixture of different glycosylated APs.

The gene for human TNAP was cloned in 1986 (Weiss, et al, 1986), Itcodes for a protein consisting of 524 amino acids with a 17 amino acidlong N-terminal signal sequence and a C-terminal GPI anchor sequencewith which the protein is anchored in viva to the outside of the plasmamembrane (Hooper, 1997). Expression of a recombinant, biologicallyactive TNAP enzyme in eukaryotic cells such as COS-1 (Fukushi, et al.,1998) and insect cells infected with baculovirus (Oda, et al., 1999) hasbeen reported.

Although discovered more than seven decades ago, the exact function ofthe TNAP molecule in bone and bone marrow tissue is unclear. Severalbiological roles for TNAP in mammals have been proposed and include:hydrolysis of phosphate esters to supply the nonphosphate moiety;transferase action for the synthesis of phosphate esters; regulation ofinorganic phosphate metabolism; maintenance of steady-state levels ofphophoxyl-metabolites; acts as a phosphoprotein phosphatase (Wbyte,1994). B/L/K-TNAP may also have a specific role in skeletalmineralization by hydrolyzing an inhibitor of calcification such asinorganic pyrophosphate, which in high concentrations can inhibit thegrowth of hydroxyapatite crystals (De Broe and Moss, 1992; Moss, 1992;Whyte, 1994). Alternatively, it has been suggested that TNAP could be aplasma membrane transporter for inorganic phosphate, an extracellularcalcium ion binding protein that stimulates calcium phosphateprecipitation and orients mineral deposition in osteoid,

TNAP is known to be a marker of osteoblast differentiation. To ourknowledge, however, there have been no previous reports of cell surfaceexpression of TNAP by immature multipotential cells.

SUMMARY OF THE INVENTION

We have recently generated a novel mAb (designated STR.O-3) thatidentifies and isolates adult multipotential cells from unfractionatedmarrow and has the capacity to subdivide the STRO-I population both invivo and in vitro. We have determined that STRO-3 binds to tissuenon-specific alkaline phosphatase (TNAP). Our results also show thatSTR.O-3 only reacts with a minor proportion of cells contained withinadult bone marrow aspirates, and does not react with CD34 positivehaemopoietic stem cells in human adult bone marrow aspixates. Thisindicates for the first time that TNAP is a marker that can be used forsingle reagent enrichment of adult multipotential cells from varioustissue sources.

Accordingly, the present invention relates to the use of TNAP as amarker for the identification and/or enrichment of adult multipotentialcells.

The present invention also provides a method of enriching for adultmultipotential cells, the method comprising preparing a cell sample froma tissue source and enriching for cells that express the TNAP marker.

In one example the method of enriching for adult multipotential cellscomprises

-   -   contacting the cell sample with a TNAP binding agent under        conditions that allows binding of TNAP to the TNAP binding        agent; and    -   separating cells bound to the TNAP binding agent.

The present invention also provides a method for identifying thepresence of an adult multipotential cell in a cell sample, the methodcomprising identifying cells in the sample that express the TNAP marker.

In one example the method for identifying the presence of adultmultipotential cells in a cell sample comprises

-   -   contacting the cell sample with a TNAP binding agent under        conditions suitable for binding of TNAP to the TNAP binding        agent; and    -   detecting the presence of the TNAP binding agent bound to cells        in the sample, wherein the presence of adult multipotential        cells is indicated by cells that bind to the TNAP binding agent

It will be appreciated that in the context of the present invention, thecell sample may be derived from any tissue source suspected ofcontaining adult multipotential cells. For example, the tissue sourcemay be adipose tissue, teeth, dental pulp, skin, liver, kidney, heart,retina, brain, hair follicles, intestine, lung, spleen, lymph node,thymus, ovary, pancreas, bone, ligament, bone marrow, tendon or skeletalmuscle. In a preferred embodiment, the tissue source is bone marrow.

The preferred source of cells is human, however, it is expected that theinvention is also applicable to other animals, including agriculturalanimals such as cows, sheep, pigs and the like, domestic animals such asdogs and cats, laboratory animals such as mice, rats, hamsters andrabbits or animals that are be used for sport such as horses.

The method may also include the harvesting of a source of themultipotential cells before the first enrichment step. This may involve,for example, surgically removing tissue from a subject and separatingthe cells of the tissue to form a single cell suspension. Thisseparation may be achieved by physical or enzymatic means. In oneexample of the invention this step involves harvesting bone marrow cellsusing known techniques.

The TNAP binding agent used in the methods of the present invention canbe any polypeptide or compound identified, as having binding affinity toTNAP. For example, the TNAP binding agent may be an, antibody orcollagen, preferably collagen type I.

The TNAP binding agent can bind to any one or mote of the LAP, KAP orBAP isoforms of TNAP. In one preferred embodiment, however, the TNAPbinding agent binds to BAP. In another preferred embodiment, the TNAPbinding agent binds specifically to BAP.

By “binds specifically to BAP” we mean that the TNAP binding agent iscapable of being bound to BAP in a selective fashion in the presence ofexcess quantities of other materials such as KAP and LAP, and tightlyenough (i.e. with high enough affinity) that it provides a useful toolfor selective enrichment of cells expressing BAP.

In a preferred embodiment, the TNAP binding agent is an anti-TNAPantibody (naturally occurring or recombinant, from any source). Theanti-TNAP antibody can be a polyclonal or monoclonal antibody. In apreferred embodiment, the anti-TNAP antibody is monoclonal antibody.

Examples of suitable anti-TNAP monoclonal antibodies for use in thepresent invention include B4-78, 50 and B4-50 (Developmental StudiesHybridoma Bank, University of Iowa); abl7973 and abl7989 (Abeam Ltd,Cambridge, UK); and the anti-TNAP mAbs referred to in Magnusson et al.(2002).

In a particularly preferred embodiment of the present invention, theanti-TNAP monoclonal antibody is a STRO-3 antibody produced by thehybridoma cell line deposited with ATCC on 19 Dec. 2005 wider theprovisions of the Budapest Treaty under deposit accession numberPTA-7282, or a mAb that binds to the same epitope on TNAP as the STR.-3antibody.

The method of enriching for adult multipotential cells according to thepresent invention may be based on the presence of the TNAP marker alone.In other words, the method of enrichment may involve a single reagent(i.e. a TNAP binding agent).

It will be understood, however, that the invention is not limited to theenrichment of cells by their expression of only TNAP, and in somecircumstances it may be preferred to enrich for adult multipotentialcells based on the expression of TNAP in combination two, three or moreadditional markers. Accordingly, the method of enriching for adultmultipotential cells may also be based on the additional presence of oneor more markers selected from the group consisting of, LFA-3, THY-I,VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, CD49a/CD49b/CD29,CD49c/CD29, CD49d/CD29, CD29, CD18, CD61, integrin beta, 6-19,thrombomodulin, CD1O, CD13, SCF, PDGF-R, EGF-R, IGF1-R, NGF-R, FGF-R,Leptin-R, (STRO-2-Leptin-R), RANKL, STRO-1 (preferably STRO-1^(bri)) andCD 146 or any combination of these markers.

For example, the method may include the step of making a first partiallyenriched pool of cells by enriching fox the expression of a first adultmultipotential cell specific marker, followed by a step of enriching forexpression of TNAP from the partially enriched pool of cells. In anotherexample, the method may include an initial enrichment step based onselection of cells expressing TNAP, followed by a step which involvesenriching for a different adult multipotential cell marker. In yetanother example, the method involves simultaneously selecting for cellsthat express TNAP and one or more additional adult multipotential cellspecific markers.

It will be understood that recognition of cells carrying TNAP that formsthe basis of the separation can be effected by a number of differentmethods, however, all of these methods rely at some point upon bindingof cells to the TNAP binding agent followed by separation of those cellsthat exhibit binding, being either high level binding, or low levelbinding or no binding. The most convenient binding agents are antibodiesor antibody based molecules, preferably being monoclonal antibodies orbased on monoclonal antibodies because of the specificity of theselatter agents.

The TNAP binding agents may be attached to a solid support to allow fora crude separation. The separation techniques preferably maximise theretention of viability of the fraction to be collected. Varioustechniques of different efficacy may be employed to obtain relativelycrude separations. The particular technique employed will depend uponefficiency of separation, associated cytotoxicity, ease and speed ofperformance, and necessity for sophisticated equipment and/or technicalskill. Procedures for separation may include, but ate not limited to,magnetic separation, using antibody-coated magnetic beads, affinitychromatography and “panning” with antibody attached to a solid matrix.Techniques providing accurate separation include but are not limited toMACS₅ Dynal magnetic bead selection and FACS-

In one example of the invention, the TNAP binding agent is labelled. Inanother example, the separatum of cells bound to the TNAP binding agentis carried out by a mechanical cell sorter.

In a further example of the invention the TNAP binding agent is coupledto a fluorescent labelling compound. In this case the separation ofcells bound to the TNAP binding agent is preferably carried out using afluorescence-activated cell sorter (FACS).

In a further example of the invention, the TNAP binding agent is linkedto a solid particle. Preferably, the solid particle is a magneticparticle. In this embodiment of the invention, the separation of cellsbound to the TNAP binding agent is preferably carried out by separatingthe particulate! phase from the liquid phase. In a further preferredembodiment of the invention, prior to the separation, step the cellsample is contacted with an antibody directed against the TNAP bindingagent linked to a solid particle, and wherein the separation of cellsbound to the TNAP binding agent is earned out by separating theparticulate phase from the liquid phase.

In a further example of the invention the cells of the cell sample areadherent cells cultivated on a solid support, and removal of unboundTNAP binding agents is carried out by rinsing.

A further example of the invention, the cells of the cell sample arecultivated in suspension, and removal of unbound TNAP binding agents iscarried out by centrifuging the cell sample and separating off theresulting supernatant

In a further example, the cell sample is subjected to a further cellsorting procedure to enrich or diminish the cell population in cellsexpressing at least one further muMpotential cell marker. Themultipotential cell marker may one or more markers selected from thegroup consisting of LFA-3, THY-I, VCAM-I, ICAM-1, PECAM-1, P-selectin,L-selectin, CD49a/CD49b/CD29, CD49c/CD29, CD49d/CD29, CD29, CD18, CD61,integrin beta, 6-19, thrombomodulin, CD1O, CD13, SCF, PDGF-R, EGF-R,IGF1-R, NGF-R, FGF-R, Leptin-R, (STRO-2=Leptin-R), RANKL, STRO-1(preferably STRO-1^(bri)) and CD146 or any combination of these markers.

The present invention also provides an enriched population of adultmultipotential cells as obtained by a method according to the presentinvention.

The present invention also provides an enriched population of TNAP+adult multipotential cells.

In a preferred embodiment of the present invention, at least 1%, 2%, 3%,4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of thetotal enriched cell population are adult multipotential cells that havethe phenotype TNAP+.

In an embodiment, culturing the enriched population of the inventionresults in a higher proportion of cells that are STRO+ when compared tocells selected using STRO-1 as a marker and cultured under the sameconditions. Preferably, such culturing is for about 4 or about 6passages. Preferably, the cells were obtained from the bone marrow.

In another embodiment, the enriched population of the inventioncomprises about 79% to about 99%, more preferably about 84% to about94%, and even more preferably about S9%, cells which are CD45+.

Preferably, the enriched population of adult multipotential cells wereobtained by a method according to the present invention.

The present invention also provides an expanded cell population obtainedby culturing an enriched population of adult multipotential cellsaccording to the invention.

In one embodiment, the enriched cell population of the invention, or anexpanded cell population of the invention, comprises at least some cellswhich are genetically modified.

The present invention also provides a method of generating a tissuespecific committed cell population, the method comprising

-   -   culturing a population of adult multipotential cells of the        present invention in the presence of one or more stimulatory        factors, and    -   subjecting said cultured population to conditions biasing        differentiation of the adult multipotential cells to a specific        tissue type.

In one embodiment of this method of the invention the tissue type isselected from the group consisting of cardiac muscle, vascular tissue,bone tissue, cartilage tissue, fat tissue, neural tissue, smooth muscleand endothelial tissue.

The invention will also be understood to encompass a compositioncomprising enriched adult multipotential cells of the present inventionand/or an expanded cell population of the invention.

In a preferred embodiment, the composition further comprises astimulatory factor. Such a composition is likely to be beneficialtherapeutically and thus will be prepared in a pharmaceuticallyacceptable form.

The level of the stimulatory &ctor(s) present in the composition may bedetermined empirically but in most cases is likely to be in the order ofnanograms or tens of nanograms per millilitre.

The stimulatory factor used in a method of the invention, and/or presentin a composition of the invention, can be any suitable factor capable ofpromoting cell division and/or differentiation. Such factors are wellknown in the art and include, but are not limited to.1α,25-dihydroxyvitamin D₃ (1,25D), platelet derived growth factor(PDGF), tumor necrosis factor α (TNF-α), interleukin-1β (TL-1β) andstromal derived factor 1α (SDF-1α).

In another embodiment the composition further comprises a factor to biasdifferentiation of the adult multipotential cells of the presentinvention to one specific tissue type. Preferably, the tissue type isselected from the group consisting of cardiac muscle, vascular tissue,bone tissue, cartilage tissue, iai tissue, neural tissue, smooth muscleand endothelial tissue

Conditions that bias differentiation of the adult multipotential cellsof the present invention to bone precursor cells or bone may involve,for example, culturing in αMEM supplemented with 10% FCS, 10% FCS, 100μM L-ascorbate-2-phosphate, dexamethasone 10⁻⁷ M and 3 mM inorganicphosphate. These conditions have been shown to induce human bone marrowstromal cells to develop a mineralized bone matrix in vitro (Gronthos etal, 1994).

Suitable conditions for differentiating the adult multipotential cellsof the present invention into osteoblasts may involve cultivating thecells in the presence of type I collagen, fibrinogen, fibrin,polyglycolic acid, polylactic acid, osteocalcin, or osteonectin. In oneparticular example, the cells are cultivated in the presence of typecollagen, fibrinogen, and fibrin. In an alternative example, the cellsare cultivated in. the presence of type I collagen, fibrinogen, fibrin,osteocalcin, and osteonectin. In the context of this method, type Icollagen, fibrinogen, fibrin, polyglycolic acid, polylactic acid,osteocalcin, or osteonectin may be used alone or in the presence of agrowth factor. It will be understood that any combination of thecompounds listed above in this paragraph is contemplated by the presentinvention.

In a further embodiment, a composition of the invention furthercomprises a fibrin glue.

The present invention also provides a method for generating or repairingtissue in a subjects the method comprising administering to the subjectan enriched or expanded cell population of the present invention.

The present invention also provides a method for generating or repairingtissue in a subject, the method comprising administering to the subjecta composition of the present invention.

The enriched or expanded cell population of multipotential cellsobtained according to the present invention may be used, for example, inthe formation and repair of bones, and as such a combination ofmultipotential cells as well as a suitable support may be introducedinto a site requiring bone formation. Thus, for example, skeletaldefects caused by bone injury or the removal of sections of boneinfected with tumour may be repaired by implanting cultured or expandedadult multipotential cells contained in calcium phosphate ceramicvehicles into the defect site. For appropriate methods and techniquessee Caplan et aL in U.S. Pat. No. 5,226,914 and U.S. Pat. No. 5,837,539,both of which use cruder preparations of stem cells when compared to thepresent invention.

In addition, the enriched cell population or composition may be used toassist in anchoring prosthetic devices. Thus, the surface of aprosthetic device such as those used in hip, knee and shoulderreplacement, may be coated with the enriched multipotential cells priorto implantation. The multipotential cells may then differentiate intoosteogenic cells to thereby speed up the process of bony ingrowth andincorporation of the prosthetic device (see Caplan. et al. in U.S. Pat.No. 5,226,914 and U.S. Pat. No. 5,837,539).

The enriched cell population or composition might also be used in genetherapy so that, for example, an enriched population may have exogenousnucleic acid transformed into it and then such a population may beintroduced into the body of the patient to treat a disease or condition.Alternatively it might be used for the release of therapeutics. Forappropriate techniques we refer to U.S. Pat. No. 5,591,625 by Gerson etal. which uses cruder preparations of stem cells when compared to thepresent invention.

Alternatively the enriched population or composition may be used toaugment bone marrow transplantation, wherein the composition containingenriched adult multipotential cells can be injected into a patientundergoing marrow transplantation prior to the introduction of the wholemarrow. In this way the rate of haemopoiesis may be increased,particularly following radiation or chemotherapy. The composition mightalso encompass a mixture of multipotential cells and haemopoietic cellswhich may be useful in radiotherapy or chemotherapy.

Also provided is the use of an enriched or expanded cell population ofthe present invention for the manufacture of a medicament for generatingor repairing tissue in a subject

Also provides is the use of a composition of the present invention forthe manufacture of a medicament for generating or repairing tissue in asubject.

The present invention also provides an isolated cell which has beenobtained by a method of the invention, or a progeny cell thereof,wherein the cell is genetically modified.

In a preferred embodiment, the cell is genetically modified to express aheterologous protein. The heterologous protein may be any protein ofinterest. For example, the heterologous protein may be a stimulatoryfactor such as 1α,25-dihydroxyvitamin D₃ (1,25D), platelet derivedgrowth factor (PDGF), tumor necrosis factor α (TNF-α), interleukin-1β(IL-1β) and stromal derived factor Ia (SDF-1α),

In another example, the heterologous protein is a bioactive factor whichaccelerates differentiation of the adult multipotential cell to specifictissue types. The bioactive factor may be, for example, a syntheticglucocorticoid, such as dexamethasone, or a bone morphogenic protein,such as BMP-2, BMP-3, BMP-4, BMP-6 or BMP-7.

The present invention also provides a [STRO-3] hybridoma cell linedeposited with ATCC on 19 Dec. 2005 under fixe provisions of theBudapest Treaty under deposit accession number PTA-7282.

The present invention also provides a STR.O-3 antibody produced by thehybridoma cell line deposited with ATCC on 19 Dec. 2005 under theprovisions of the Budapest Treaty under deposit accession numberPTA-7282.

The present invention also provides an isolated antibody which binds tothe same epitope on multipotential cells as the STRO-3 antibody producedby the hybridoma cell line deposited with ATCC on 19 Dec. 2005 under theprovisions of the Budapest Treaty under deposit accession numberPTA-7282.

The present invention also provides a composition comprising an antibodyof the invention. Preferably, the composition further comprises one ormore suitable carriers.

Also provided is a kit comprising an enriched cell population of theinvention, an expanded cell population of the invention, a compositionof the invention, an isolated cell of the invention, a hybridoma of theinvention and/or an antibody of the invention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The various features and embodiments of the present invention, referredto in individual sections above apply, as appropriate, to othersections, mutatis mutandis.

Consequently features specified in one section may be combined withfeatures specified in other sections, as appropriate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Flow Cytometric Analysis of STRO-3-Sebcted BAF-3 Cells

Progressive enrichment of BAF-3 expressing the STRO-3 surface Ag. BAF-3cells selectively isolated by the magnetic bead/mAb capture andenrichment procedure were immunolabeled with the STRO-3 mAb and analyzedby flow cytometry after one (A), two (B), and three (C) rounds ofselection. Magnetic bead selection and enrichment were carried out untilhomogeneity of Ag expression was achieved.

FIG. 2. PCR recovery of proviral cDNA inserts from genomic DNA isolatedfrom BAF-3 cells expressing STRO-3 surface Ag

Long range PCR was used to recover the cDNA inserts from genomic DNA(arrow) isolated from the BAF-3 cells expressing the STRO-3 cell surfaceAg. The PGR primers used were complementary to the sequences adjacent tothe multi-do πing site in the retroviral vector. Amplification wasperformed as detailed in Methods, after which the PCR products wereseparated on a 1.0% agarose gel and visualised by ethidium bromidestaining.

FIG. 3. FASTA Alignment Analysis of STRO-3 antigen derived PCR Products

Following partial sequence analysis, the resultant nucleotide sequencewas compared with sequences submitted to the combined Geribank/EMBLdatabase via standard “FASTA alignment analysis”, and revealed 100%homology with the BLK isoform of ALP complementary DNA sequence (Genbankaccession #37944).

FIG. 4. STRO-3 mAb recognise the BLK isoform of ALP in BAF-3Transfectants

A 1.7 kb BamH-hoII restriction fragment of the BLK-ALP cDNA (harbouringboth the entire coding sequence and the 5′ and 3′ non-coding regions)was subcloned into the pR_F.

o vector and subsequently introduced into BAF-3 cells by retroviraltransduction (refer to Materials and Methods). The resultantG418-resistant cell population, was stained by indirectimmunofluorescence and analysed by flow cytometry. Data are displayed assingle-parameter fluorescence (FITC) histograms of 1×10⁴ light-scattergated events, collected as list mode data IgG1 control (thin blackline); (A) mAb STRO-3; (B) mAb B4-78; (C) mAb B4-50 and (d) mAb 8B6.

FIG. 5. STRO-3 mAb Identifies an Enzymatically Active Form of ALP

Cytospia preparations of untransfected (A) and STRO-3 positive (B) BAF-3cells were prepared on glass slides then fixed with 70% ethanol. Theslides were then incubated with alkaline phosphatase substrate using theSigma Alkaline Phosphatase Substrate Kit (AMOIOO) as recommended by themanufacturer. The results showed that BAF-3 cells expressing STRO-3 (B)contained the active form of alkaline phosphatase enzyme (purple/redcolour). The cells were counter stained with Haematoxylin (blue).

FIG. 6. ALP specific PCR

RT-PCR was employed to identify the alkaline phosphatase isoform encodedby the cDNA using total RNA isolated from BAF-3 cells expressing theSTRO-3 cell surface Ag as described in the methods. The PCR primers usedidentified sequences specific to either the (L) liver (216 bp) or (B)bone (215 bp) alkaline phosphatase isoforms as previously described bySato and colleagues (1994) (Sato et al., 1994). Following PCR amplifcation the products were run on a 1.5% agarose gel and stained withethidium bromide. The results indicated that the STRO-3 antigenexpressing BAF-3 cells only expressed transcripts corresponding to thebone-specific (B) alkaline phosphatase isoform.

FIG. 7. The Expression of STRO-3 Antigen in Human Bone Tissue

The immunoreactivity of STRO-3 mAb was also assessed in sections ofdeveloping bone marrow using immunohistochemistry as described in themethods. Five micron sections of paraffin-embedded, 55 day old humanlimb, was stained with STRO-3 mAb, as described in the methods. Whileexpression of the STRO-3 antigen (TNAP) was evident in the mesenchymalcells of the bone marrow spaces (BM), perivascular regions (PV) and atthe interface of the growth plate region, no staining was observed inthe periosteum (P) or cartilage (C).

FIG. 8. Clonogenic cells are Exclusively Restricted to the STRO-3 mAbPositive Fraction of Human BM

A single cell suspensions of unfractionated BM (Pre) and MACS selectedTNAP positive (TNAP+) and TNAP negative (TNAP−) human BM were platedinto regular growth medium (Gronthos et al., 2003) to assess theincidence of adherent colony-forming cells in each cell fraction.Following 12 days of culture, colonies (aggregates of 50 cells or more)were stained and, scored as described in Methods. The bar graph depictsthe number of elenaogenic colonies per 10⁵ cells plated for each cellfraction averaged from two separate experiments. Our data demonstratethat CFU-F are exclusively restricted to the TNAP positive fraction ofBM.

FIG. 9. Co-expression of TNTAP and the Mesenchymal Precursor CellMarker, STRO-1 by Adult Human BMMNC

Dual-color immunofluorescence and flow cytometry was performed byincubation of STRO-I MACS-selected BMMNC and indirectly labelled with agoat anti-murine IgM antibody coupled to FITC (x axis), and STRO-3 mAb(murine IgG1) indirectly labelled with a goat anti-murine IgG coupled toPE (y axis). The dot plot histogram represents 5×10⁴ events collected aslistmode data. The vertical and horizontal lines were set to thereactivity levels of <1.0% mean fluorescence obtained with theisotype-matched control antibodies, 1B5 (TgG) and 1A6. 12 (IgM) treatedunder the same conditions. The results demonstrate that a minorpopulation, of STRO-1 bright cells co-expressed TNAP (upper rightquadrant) while the remaining STRO-1+ cells failed to react with theSTRO-3 mAb. Cells isolated by FACS from all four quadrants weresubsequently assayed for the incidence of CFU-F (Table 2).

FIG. 10. Compression of CD45, CD34, 30%, CC9 and STRO-I with TNAP bySTR.0-3 mAb enriched cells

FIG. 11. STRO-3 mAb selected cells maintain high levels of STRO-Iexpression following multiple passages

FIG. 12. STRO-1 expression in bone marrow derived cells selected usingan antibody that binds thereto

FIG. 13. Early (P2) and late (P5) passage phenotypic characteristics ofSTRO-3 mAb selected, culture expanded muMpotential cells

STRO-3 selected adult multipotential cells (P1) are a population with asurface phenotype characterised by high levels of CC9, STRO-1 and STRO-3antigen expression. Following 5 passages in culture the cell population(P5) demonstrates significant retention of STRO-1 expression.

FIG. 14. Differentiation of STRO-3 mAb selected cells into adipocytes

Two lots of STRO-3 _(maB)Ab selected cells, 2242A and 2070C, wereassayed for differentiative capacity. The graphs depict the averagerelative fluorescence units (Avg RFU) for cells induced with AdipogenicInduction Medium versus control uninduced cells.

FIG. 15. Differentiation of STRO-3 mAb selected cells into osteocytes

A standard curve was generated by taking the OD 550nr α of sampleshaving known calcium concentrations. Two lots of STRO-3 _(mAb)Abselected cells, 2242A and 2070C, were induced with OsteogenesisInduction Medium. Cell extracts of induced and uninduced cells wereprepared and measured at OD 550 nm.

FIG. 16. Differentiation of STRO-3 mAb selected cells into functionalosteoblasts

A—STRO-3 mAb selected adult multipotential cells were cultured for threeweeks in

EM supplemented with 10% FCS, 100 μM L-ascorbate-2-phosphate,dexamethasone 10-7 M and 3 mM inorganic phosphate and stained formineral deposits with Alizarin Red. B—Oil Red O stained cells followingculture of STRO-3 mAb selected adult multipotential cells in thepresence of 0.5

M methylisobutylmethylxanthine, 0.5 μM hydrocortisone, and 60 μMindomethacin. C—Cell cultures treated with 10 ng/ml TGF-p3 and stainedwith Alcian Blue to identify proteoglycan synthesis. D—Histologicalexamination of culture expanded STRO-3 mAb selected adult multipotentialcells following implantation.

FIG. 17. Rate of spinal fusion following administration of cultureexpanded STRO-3 mAb selected cells

FIG. 18. Robust spinal fusion in culture expanded STRO-3 mAb selectedcells treated sheep

FIG. 19. STRO-3 mAb selected culture expanded allogeneic adultmultipotential cells in an ovine transpedicular screw fixation model

FIG. 20. Dose-dependent bone growth by allogeneic culture expandedSTRO-3 mAb selected cells in critical-sized sheep segmental tibialdefect

FIG. 21. Greater rate of union in culture expanded STRO-3 mAb selectedcells treated groups with critical sized segmental tibial defect

FIG. 22. Culture expanded STRO-3 mAb selected cells significantlyimprove cardiac function 2 weeks following rat myocardial infarction

FIG. 23. Effects of allogenic sheep culture expanded STRO-3 selectedcells (passage 5) directly injected into sheep heats immediately afteracute ligation of both diagonal and coronary arteries

A—% reduction in ejection fraction, B—% change in diastolic volume, andC—% change in systolic volume of sheep.

FIG. 24. Increased cell survival when delivered in fibrin glue comparedto saline solution

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Micro-Organism DepositDetails

The hybridoma which produces the monoclonal antibody designated STRO-3was deposited on 19 Dec. 2005 with American Type Culture Collection(ATCC) under accession number PTA-7282.

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder. This assuresmaintenance of viable cultures for 30 years from the date of deposit.The organisms will be made available by ATCC under the terms of theBudapest Treaty which assures permanent and unrestricted availability ofthe progeny of the culture to the public upon issuance of the pertinentpatent

The assignee of the present application has agreed that if the culturedeposit should die or be lost or destroyed when cultivated undersuitable conditions, it will be promptly replaced on notification with aviable specimen of the same culture. Availability of a deposited strainis not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

General Techniques

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken o have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, immunology, intmunohistochemistry, proteinchemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature is sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John. Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub,Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988); and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

Adult Multipotential Cells

By “adult multipotential cells” we mean cells derived from adult tissuewhich are capable of giving rise to any of several mature cell types. Asused herein, this phrase encompasses adult stem cells and progenitorcells, such as mesenchymal precursor cells (MPC) and multipotentialprogeny of these cells.

Mesenchymal precursor cells (MPCs) are cells found in bone marrow,blood, dermis, and periosteum; and are capable of differentiating intospecific types of mesenchymal or connective tissues including adipose,osseous, cartilaginous, elastic, muscular, and fibrous connectivetissues. The specific lineage-commitment and differentiation pathwaywhich these cells enter depends upon various influences from mechanicalinfluences and/or endogenous bioactive factors, such as growth factors,cytokines, and/or local microenvironment conditions established by hosttissues. Mesenchymal precursor cells are defined as cells which are notterminally differentiated; which can divide without limit; and divide toyield daughter cells that ate either stem cells or are progenitor cellswhich in time will irreversibly differentiate to yield a phenotypiccell. MPCs are non-hematopoietic progenitor cells that are capable offorming large number of multipotential cells.

The terms ‘enriched’, ‘enrichment’ or variations thereof are used hereinto describe a population of cells in which the proportion of oneparticular cell type or the proportion of a number of particular celltypes is increased when compared with the untreated population.

In a preferred embodiment of the present invention, at least 1%, 2%, 3%,4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of thetotal enriched cell population are adult multipotential cells that havethe phenotype TNAP+.

In a particularly preferred embodiment, TNAP+ cells of the invention areable to bind the STRO-3 antibody produced by the hybridoma cell linedeposited with ATCC on 19 Dec. 2005 under the provisions of the BudapestTreaty under deposit accession number PTA-7282.

In one embodiment, the enriched population of the invention comprisesabout 79% to about 99%, more preferably about 84% to about 94%, and evenmore preferably about 89.2%, cells which are CD45+.

In another embodiment, the enriched population of the inventioncomprises less than about 2%, more preferably less than about 1%, cellsthat are CD34+. In another embodiment, the enriched population comprisesno cells that are CD34+.

In another embodiment, the enriched population of the invent oncomprises less than about 6%, more preferably less than about 3.5%,cells that are CC9+.

La a further embodiment, the enriched population of the inventioncomprises about 23% to about 3%, more preferably about 8% to about 18%,and even more preferably about 13.2%, cells which are 3G5+.

In yet a further embodiment, the enriched population of the inventioncomprises about 12% to about 3%, more preferably about 10% to about 6%,and even more preferably about 7.8%, cells which are STRO-1+.

In a further embodiment, an enriched cell population of the inventionhas not been cultured in vitro.

Furthermore, in a preferred embodiment, the enriched cell population ofthe invention is capable of giving rise to clonogenic CFU-F.

In an embodiment, culturing the enriched population of the inventionresults in a higher proportion of cells that are STR.O+ when compared tocells selected using STRO-1 as a marker and cultured under the sameconditions. Preferably, such culturing is for about 4 or about 6passages. Preferably, the cells were obtained from the bone marrow.

In a further embodiment, culturing the enriched population of theinvention results in an increase in the number of progeny cells mat areSTRO+ when compared to the starting cell population, for example, after2, 4 or 6 passages. In comparison, culturing STRO-1 enriched cellsresults in a decreased number of progeny cells that are STRO+ whencompared to the starting (STRO-1 selected) cell population, for example,after 4 or 6 passages.

In another embodiment, the enriched cell population is homogenous forTNAP+ cells,

The present invention also relates to progeny cells (also referred toherein as expanded cells) which are produced from the in vitro cultureof adult multipotential cells of the invention. Expanded cells of theinvention may a have a wide variety of phenotypes depending on theculture conditions (including the number and/or type of stimulatoryfactors in the culture medium), the number of passages and the like.

In one embodiment, such expanded cells (at least after 5 passages) canbe TNAP−, CC9⁺, HLA class 1, HLA class IF, CD14″, CD 19″, CD3″,CD11a-c″, CD31″, CD86″ and/or CD 80″. However, ft is possible that underdifferent culturing conditions to those described herein that theexpression of different markers may vary. Also, whilst cells of thesephenotypes may predominate in the expended cell population it does notmean that there is a minor proportion of the cells do not have thisphenotype(s) (for example, a small percentage of the expanded cells maybe CC9−). In one preferred embodiment, expanded cells of the inventionstill have the capacity to differentiate into different cell types.

In one embodiment, an expended cell population of the inventioncomprises cells wherein at least 25%, more preferably at least 50%, ofthe cells are CC9+.

In another embodiment, an expended cell population of the inventioncomprises cells wherein at least 40%, more preferably at least 45%, ofthe cells are STRO-1+.

In, a further embodiment, culturing the enriched population of theinvention results in adult multipotential cells that may also expressmarkers selected from the group consisting of LFA-3, THY-I, VCAM-1,ICAM-1, PECAM-1, P-selectin, L-selectin, CD49a/CD49b/CD29, CD49c/CD29,CD49d/CD29, CD29, CD 18, CD61, integrin beta, 6-19, thrombomodulin,CD10, CD13, SCF, PDGF-R, EGF-R

, IGF1-R, NGF-R, FGF-R Leptin-R, (STRO-2=Leptin-R), RANKL,STRO-1^(bright) and CD146 or any combination of these markers.

It is preferred that a significant proportion of the adultmultipotential cells are capable of differentiation into at least twocommitted cell types. Non-limiting examples of the lineages to which theadult multipotential cells may be committed include bone precursorcells; hepatocyte progenitors, which are pluripotent for bile ductepithelial cells and hepatocytes; neural restricted cells, which cangenerate glial cell precursors that progress to oligodendrocytes andastrocytes; neuronal precursors that progress to neurons; precursors forcardiac muscle and cardiomyocytes, glucose-responsive insulin secreti Ogpancreatic beta cell lines. Other lineages include, but are not limitedto, odontoblasts, dentfrvproducing cells and chondrocytes, and precursorcells of flie following: retinal pigment epithelial cells, fibroblasts,skin cells such as kerathiocytes, dendritic cells, hair follicle cells,renal duct epithelial cells, smooth and skeletal muscle cells,testicular progenitors, vascular endothelial cells, tendon, ligament,cartilage, adipocyte, fibroblast, marrow stroma, cardiac muscle, smoothmuscle, skeletal musole, pericyte, vascular, epithelial, glial,neuronal, astrocyte and oligodendrocyte cells. In a preferredembodiment, the adult multipotential cells are at least capable of beingcultured, in vivo or in vitro, to produce adipocytes, osteocytes and/orchondrocytes.

In another embodiment, “adult multipotential cells” of the invention arenot capable of giving rise, upon culturing, to hematopoietic cells.

The term “adult” is used in its broadest sense to include a postnatalsubject. In a preferred embodiment, the term “adult” refers to a subjectthat is postpubertal. The term, “adult” as used herein can also includecord blood taken from a female. The term “adult” does not include cellsobtained from an embryo and/fetus. Thus, the “adult multipotentialcells” of the invention may also be considered as “non-embryonicmultipotential cells”.

When we refer to a cell as being “positive” for a given marker it may beeither a low (Io or dim) or a high (bright bri) expresser of that markerdepending on the degree to Which the marker is present on the cellsurface, where the terms relate to intensity of fluorescence or othercolour used in the colour sorting process of the cells: The distinctionof Io (or dim or dull) and bri will be understood in the context of themarker used on a particular cell population being jsorted. When we referherein to a cell as being “negative” for a given marker, it does notmean that the marker is not expressed at all by that cell. It means thatthe marker is expressed at a relatively very low level by that cell, andthat it generates a very low signal when detectably labelled.

The term “bright”, when used herein, refers to a marker on a cellsurface that generates a relatively high signal when detectablylabelled. Whilst not wishing to be limited by theory, it is proposedthat “bright” cells express more of the target marker protein (forexample the antigen recognised by STRO-I) than other cells in thesample. For instance, STRO-1^(bri) cells produce a greater fluorescentsignal, when labelled with a FITC-conjugated STRO-1 antibody asdetermined by FACS analysis, than non-bright cells (STRO-1^(dull/dim)).Preferably, “bright” cells constitute at least about 0.1% of the mostbrightly labelled bone marrow mononuclear cells contained in thestarting sample, In other embodiments, “bright” cells constitute atleast about 0.1%, at least about 0.5%, at least about 1%, at least about1.5%, or at least about 2%, of the most brightly labelled bone marrowmononuclear cells contained in the starting sample. In a preferredembodiment, STRO-1^(bright) cells have 2 log magnitude higher expressionof STRO-I surface expression. This is calculated relative to“background”, namely cells that are STRO-F. By comparison, STRO-1^(dim)and/or STRO-1^(intermediate) cells have less than 2 log magnitude higherexpression of STRO-1 surface expression, typically about 1 log or lessthan “background”.

Tissue Non-Specific Alkaline Phosphatase (TNAP)

When used herein the term “TNAP” is intended to encompass all isofoπnsof the protein. For example, the term encompasses the liver isoform(LAP), the bone isoform (BAP) and the kidney isoform (KAP). In apreferred embodiment, the TNAP is BAP. La a particularly preferredembodiment. TNAP as used herein refers to a molecule which can bind theSTRO-3 antibody produced by the hybridoma cell line deposited with ATCCon 19 Dec. 2005 under the provisions of the Budapest Treaty underdeposit accession number PTA-7282.

Is the context of the present invention, the TNAPP is preferably humanTNAP. For example, the TNAP may be human TNAP comprising the amino acidsequence shown in SEQIDNO:1.

However, it will be understood that the term “TNAP” is not limited tothe human sequence but also includes homologous sequences obtained fromany source, for example homologues, particularly orthologues (i.e.homologues obtained from species other than humans), allelic variants,as well as fragments and synthetic peptides or derivatives thereof asdiscussed below.

A number of TTNAP orthologues are already known and include mouse TNAP(SEQ ID NO:2) and rat TNAP (SEQ ID NO:3).

In a preferred embodiment of the present invention, a homologoussequence is an amino acid sequence which is at least 70, 80 or 90%identical, preferably at least 95 or 98% identical at the amino acidlevel over at least 20, preferably 50 or 100 amino acids with a sequenceas shown SEQ ID NO:1, SEQ ID NO;2 or SEQ ID NO:3.

Although homology can also be considered in the art in terms ofsimilarity (i.e. amino acid residues haying similar chemicalproperties/functions), in the context of the present invention it ispreferred to express homology in terms of sequence identity. The %identity of a polypeptide Js determined by FASTA (Pearson and Lipman,1988) analysis (GCG program) using the default settings and a querysequence of at least 50 amino acids in length, and whereby the FASTAanalysis aligns the two sequences over a region of at least 50 aminoacids. More preferably, the FASTA analysis aligns the two sequences overa region of at least 100 amino acids. Moxe preferably, the FASTAanalysis aligns the two sequences over a region of at least 250 aminoacids. Even more preferably, the FASTA analysis aligns the two sequencesover a region of at least 350 amino acids,

The terms “variant” or “derivative” in relation to the amino acidsequences of the present invention and/or for use in the presentinvention includes any substitution of, variation of, modification of,replacement of, deletion of or addition of one (or more) amino acidsfrom or to the sequence providing the resultant amino acid sequencepreferably has at least 25 to 50% of the biological activity as anaturally occurring TNAP more preferably at least substantially the sameactivity. The relevant biological activity includes the ability of thevariant or derivative to bind to natural TNAP ligands.

In general, preferably less than 20%, 10% or 5% of the amino acddresidues of a variant or derivative are altered as compared with thecorresponding region of the naturally occurring TNAP, thepercentage-typically being lower the shorter the amino, acid sequencee.g. less than 5% for amino acid sequences of 20 amino acids or less.

The term “TNAP” also encompasses fragments of the above mentionedfull-length polypeptides and variants thereof, including fragments ofthe sequences set out in the sequence listing herein. Preferredfragments include those that include an epitope. Suitable fragments willbe at least about 6 or 7 amino acids in length, e.g. at least 10, 12, 15or 20 amino acids in length. They may also be less than 200, 100 or 50amino acids in length. Polypeptide fragments of the polypeptidesdepicted in the sequence listings and allelic and species variantsthereof may contain one or more (e.g. 2, 3, 5, or 10) substitutions,deletions or insertions, including conserved substitutions. Wheresubstitutions, deletion and/or insertions have been made, for example bymeans of recombinant technology, preferably less than 20%, 10% or 5% ofthe amino acid residues depicted in the sequence listings are altered.

In an embodiment, the term TNAP does not encompass placental AP.

TNAP Binding Agents

When used herein, the phrase “TNTAP binding agent” refers to a moietythat recognises and/or binds to TNAP.

Preferred TNAP binding agents are polypeptides or compounds identifiedas having binding affinity to TNAP. For example,

TNAP has been characterised as having a collagen binding loop (Mornet etal., 2001). Accordingly, the TNAP agent may be collagen, preferably typeI collagen.

Particularly preferred TNAP binding agents are anti-TNAP antibodies(naturally occurring or recombinant, from any source).

The term “antibody” as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab′)2, and Fv which arecapable of binding the epitopic determinant These antibody fragmentsretain some ability to selectively bind with its antigen or receptor andare defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chains two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)2, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab)2 is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbour Laboratory. New York (1988), incorporated herein by reference).

Antibodies of the present invention can be prepared using cellsexpressing TNAP, full length TNAP or fragments thereof as the immunizingantigen. A peptide used to immunize an animal can be derived fromtranslated cDNA or chemical synthesis and is purified and conjugated toa carrier protein, if desired. Such commonly used carriers which arechemically coupled to the peptide include keyhole limpet hemocyanin(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.The coupled peptide may then be used to immunise the animal (e.g., amouse or a rabbit).

If desired, polyclonal antibodies can be further purified, for example,by binding to and ehition from a matrix to which the peptide to whichthe antibodies were raised is bound. Those of skill in the art will knowof various techniques common in the immunology arts for purificationand/or concentration of polyclonal antibodies, as well as monoclonalantibodies (See for example, Coligan, et al., Unit 9, Current Protocolsin Immunology, Wiley Interscience, 1991, incorporated by reference).

Monoclonal antibodies may be prepared using any technique which providesfor the production of antibody molecules by continuous cell lines inculture, such as, for example, the hybridoma technique, the human B-cellhybridoma technique, and the EBV-hybiidoma technique (Kohler et al.,1975; Kozbor et al., 1985; Cote et al., 1983; Cole et al., 1984).

Methods known in the art also allow antibodies exhibiting binding forTNAPP to be identified and isolated from antibody expression libraries.

Antibodies with an epitopic specificity which is the same as or similarto that of mAb STRO-3 can be identified by their ability to compete withthat particular mAb for binding to TNAP (e.g. to cells bearing TNAP,such as MPCs, or to isolated TNAP protein or fragments thereof). Usingreceptor chimeras (Rucker et al., 1996) or other techniques known tothose skilled in the art, the binding site of STRO-3 mAb may be mapped.

It is also possible to determine, without undue experimentation, if amonoclonal antibody has the same specificity as STRO-3 mAb byascertaining whether the former prevents the latter from binding toTNAP. If the monoclonal antibody being tested competes with STRO-3 mAb,as shown by a decrease in binding by STRO-3 mAb, then the two monoclonalantibodies bind to the same, or a closely related, epitope.

Still another way to determine whether a monoclonal antibody has thespecificity of STRO-3 mAb is to pre-incubate the monoclonal antibodybeing tested with TNAP, and then add STRO-3 mAb to determine if STRO-3mAb is inhibited in its ability to bind to TNAP. If the binding ofSTRO-3 mAb is inhibited then, in all likelihood, the monoclonal antibodybeing tested has the same, or functionally equivalent, epitopicspecificity as STRO-3 mAb.

Monoclonal antibodies useful in the present invention can be engineeredso as to change the isotype of the antibody. Fox example, an IgG2Aisotype can be engineered as an IgG1, IgG2B, or other isotypes.

It will be appreciated that a TNAP binding agent such as an antibody ofthe invention may be conjugated to a compound that is useful, forexample, in celj separation, therapeutic or diagnostic applications. Inone example, an antibody of the invention is conjugated to a label. Thelabel may be any entity the presence of which can be readily detected.For example, the label may be a direct label, such as those described indetail in May et. al., U.S. Pat. No. 5,656,503. Direct labels areentities which, in their natural state, are readily visible either tothe naked eye, or with the aid of an optical filter and/or appliedstimulation, e.g. UV light to promote fluorescence. Examples includeradioactive, chemiluminescent, electroactive (such as redox labels), andfluorescent compounds. Direct particulate labels, such as dye sols,metallic sols (e.g. gold) and coloured latex particles, are also verysuitable and are, along with fluorescent compounds, preferred. Of theseoptions, coloured latex particles and fluorescent compounds are mostpreferred. Concentration of the label into a small zone or volume shouldgive rise to a readily detectable signal, e.g. a strongly coloured area.Indirect labels, such as enzymes, e.g. alkaline phosphatase andhorseradish peroxidase, can also be us.ed, although these usuallyrequire the addition of one or more developing reagents such assubstrates before a visible signal can be detected.

Conjugation of a label to a binding agent such as an antibody of theinvention can be by covalent or non-covalent (including hydrophobic)bonding, or by adsorption. Techniques for such conjugation arecommonplace in the art and may be readily adapted for the particularreagents employed.

A binding agent for use in the methods of the invention, such as anantibody of the invention, may also be coated onto a solid support. Forexample, the antibody can be coated on, a synthetic plastics material,magnetic, particle, microtitre assay plate, microarray chip, latex bead,filter comprising a cellulosic or synthetic polymeric material, glass orplastic slide, dipstick, capillary fill device and the like.

A binding agent for use in the methods of the invention, such as anantibody of the invention, may also be incorporated into a device forcell separation. For example, the device may be an automated cellselection device based on MACS technology. Such a device enables largescale magnetic cell selection in a closed and sterile system. Forexample, the device may comprise an integrated computer, a magneticseparation unit, a peristaltic pump and various pinch valves. Theintegrated computer preferably controls all components of the instrumentand directs the system to perform procedures in a standard sequence. Themagnetic separation unit preferably includes a movable permanent magnetand a holder for the selection column. The peristaltic pump preferablycontrols the flow rate through the tubing set. Pinch valves can be usedto ensure controlled flow of buffer and cell suspension. Beforeselection the cells are magnetically labeled by using an antibody of thepresent invention. A single-use tubing set, including separationcolumns, may then be attached to the device and the cell preparationbag, containing the labeled cells-, may be connected to the tubing set.After starting the selection program, the system automatically appliesthe cell sample to the separation column, performs a series of washingsteps depending on the program chosen and finally elutes the purifiedtarget cells.

Cell-Sorting Techniques

The ability to recognise adult multipotential cells with TNAP bindingagents, such as anti-TNAP antibodies, allows not only for theidentification and quantification of these cells in tissue samples, butalso for their separation and enrichment in suspension. This can beachieved by a number of cell-sorting techniques by which cells arephysically separated by reference to a property associated with thecell-antibody complex, or a label attached to the antibody. This labelmay be a magnetic particle or a fluorescent molecule. The antibodiesmay-be cross-linked such that they form aggregates of multiple cells,which are separable by their density. Alternatively the antibodies maybe attached to a stationary matrix, to which the desired cells adhere.

Various methods of separating antibody-bound cells from unbound cellsare known. For example, the antibody bound to the cell (or an.anti-isotype antibody) can be labelled and then the cells separated by amechanical cell sorter that detects the presence of the label.Fluorescence-activated cell sorters are well known in. the art. In oneembodiment, the anti-TNAP antibody is attached to a solid support.Various solid supports are known to those of skill in the art,including, but not limited to. agarose beads, polystyrene beads, hollowfiber membranes, polymers, and plastic petri dishes. Cells that arebound by the antibody can be removed from the cell suspension by simplyphysically separating the solid support from the cell suspension.

Super paramagnetic microparticles may be used for cell separations. Forexample, the microparticles may be coated with anti-TNAP antibodies. Theantibody-tagged, super paramagnetic microparticles may then be incubatedwith a solution containing the cells of interest. The microparticlesbind to the surfaces of the desired adult multipotential cells, andthese cells can then be collected in a magnetic field.

In another example, the cell sample is allowed to physically contact,for example, a solid phase-linked anti-TNAP monoclonal antibody. Thesolid-phase linking can comprise, for instance, adsorbing the antibodiesto a plastic, nitrocellulose, or other surface. The antibodies can alsobe adsorbed on to the walls of the large pores (sufficiently large topermit flow-through of cells) of a hollow fiber membrane. Alternatively,the antibodies can be covalently linked to a surface or bead, such asPharmacia Sepharose 6 MB macrobeads. The exact conditions and durationof incubation for the solid phase-linked antibodies with the adultmultipotential cell containing suspension will depend upon severalfactors specific to the System employed. The selection of appropriateconditions, however, is well within the skill of the art.

The unbound cells are then eluted or washed away with physiologic bufferafter allowing sufficient time for the adult multipotential cells to bebound. The unbound cells can be recovered and used for other purposes ordiscarded after appropriate testing has been done to ensure that thedesired separation had been achieved. The bound cells are then separatedfrom the solid-phase by any appropriate method, depending mainly uponthe nature of the solid phase and the antibody. For example, bound cellscan be eluted from a plastic petri dish by vigorous agitation.Alternatively, bound cells can be eluted by enzymatically “nicking” ordigesting an enzyme-sensitive “spacer” sequence between the solid phaseand the antibody. Spacers bound to agarose beads are commerciallyavailable from, for example, Pharmacia.

The eluted, enriched fraction of cells may then be washed with a bufferby centrifugation and either said enriched fraction or the unboundfraction may be cryopreserved in a viable state for later use accordingto conventional technology or introduced into the transplant recipient

Production of Genetically Modified Cells

In one embodiment the present invention relates to genetically modifiedcells, particularly genetically modified adult multipotential cells ofthe invention. Preferably, the cells are genetically modified to producea heterologous protein. Typically, the cells will be geneticallymodified such that the heterologous protein is secreted from the cells.However, in an embodiment the cells can be modified to express afunctional non-protein encoding polynucleotide such as dsRNA (typicallyfor RNA silencing), an antisense oligonucleotide or a catalytic nucleicacid (such as a ribozyme or DNAzyme).

Genetically modified cells may be cultured in the presence of at leastone cytokine in an amount sufficient to support growth of the modifiedcells. The genetically modified cells thus obtained may be usedimmediately (e.g., in transplant), cultured and expanded in vitro, orstored for later uses. The modified cells may be stored by methods wellknown in the art, e.g., frozen in liquid nitrogen.

Genetic modification as used herein encompasses any genetic modificationmethod which involves introduction of an exogenous or foreignpolynucleotide into an adult multipotential cell or modification of anendogenous gene within adult multipotential cell. Genetic modificationincludes but is not limited to transduction (viral mediated transfer ofhost DNA from a host or donor to a recipient, either in vitro or invivo), transfection (transformation of cells with isolated viral DNAgenomes), liposome mediated transfer, electroporation, calcium phosphatetransfection or coprecipitation and others. Methods of transductioninclude direct co-culture of cells with producer cells (Bregni et al.,1992) or culturing with, viral supernatant alone with or withoutappropriate growth factors and polycations (Xu et al., 1994).

An exogenous polynucleotide is preferably introduced to a host cell in avector. The vector preferably includes the necessary elements for thetranscription and translation of the inserted coding sequence. Methodsused to construct such vectors are well known in the art. For example,techniques for constructing suitable expression vectors are described indetail in Sambrobk et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, N.Y. (3rd Ed., 2000); and Ausuhel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., New York(1999).

Vectors may include but are not limited to viral vectors, such asretroviruses, adenoviruses, adeno-associated viruses, and herpes simplexviruses; cosmids; plasraid vectors; synthetic vectors; and otherrecombination vehicles typically used in the art. Vectors containingboth a promoter and a cloning site into which a polynucleotide can beoperatively linked are well known in the art. Such vectors are capableof transcribing RNA in vitro or in vivo, and are commercially availablefrom sources such as Stratagene (La Jolla, Calif.) and Promega Biotech(Madison, Wis.). Specific examples include, pSG, ρSV2CAT, pXtl fromStratagene; and pMSG, pSVL, pBPV and ρSVK3 from Pharmacia.

Preferred vectors include retroviral vectors (see, Coffin et sL,“Retroviruses”, Chapter 9 pp; 437-473, Cold Springs Harbor LaboratoryPress, 1997). Vectors useful in the invention can be producedrecombinantly by procedures well known in the art. For example.WO94/29438, WO97/21824 and WO97/21825 describe the construction ofretroviral packaging plasmids and packing cell lines. Exemplary vectorsinclude the pCMV mammalian expression vectors, such as pCMV6b and pCMVGc(Chiron Corp.), pSFFV-Ne θ, and pBluescript-Sk+. Non-limiting examplesof useful retroviral vectors are those derived from murine, avian orprimate retroviruses. Common retroviral vectors include those based onthe Moloney murine leukemia virus (MoMLV-vector). Other MoMLV derivedvectors include, Lmily, LINGFER, MINGFR and MINT. Additional vectorsinclude those based on Gibbon ape leukemia virus (GALV) and Moloneymurine sarcoma virus (MOMSV) and spleen focus forming virus (SFFV).Vectors derived from the murine stem cell virus (MESV) includeMESV-MiLy. Retroviral vectors also include vectors based onlentiviruses, and non-limiting examples include vectors based on humanimmunodeficiency virus (HIV-I and HTV-2).

In producing retroviral vector constructs, the viral gag, pol and envsequences can be removed from the virus, creating room for insertion offoreign DNA sequences. Genes encoded by foreign DNA are usuallyexpressed under the control a strong viral promoter in the long terminalrepeat (LTR). Selection of appropriate control regulatory sequences isdependent on the host cell used and selection is within the skill of onein the art. Numerous promoters are known in addition to the promoter ofthe LTR, Non-limiting examples include the phage lambda PL promoter, thehuman cytomegalovirus (CMV) immediate early promoter; the U3 regionpromoter of the Moloney Murine Sarcoma Virus (MMSV), Rous Sacroma Virus(RSV), or Spleen Focus Forming Virus (SFFV); Granzyme A promoter; andthe Gxanzyτπe. B promoter. Additionally inducible or multiple controlelements may be used. The selection of a suitable promoter will beapparent to those sldlled in the art.

Such a construct can be packed into viral particles efficiently if thegag, pol and env functions are provided in trans by a packing cell line.Therefore, when the vector construct is introduced into the packagingcell, the gag-pol and env proteins produced by the cell, assemble withthe vector RNA to produce infectious virons that are secreted into theculture medium. The virus thus produced can infect and integrate intothe DNA of the target cell, but does not produce infectious viralparticles since it is lacking essential packaging sequences. Most of thepacking cell lines currently in use have been transfected with separateplasrπids, each containing one of the necessary coding sequences, sothat multiple recombination events are necessary before a replicationcompetent virus can be produced. Alternatively the packaging cell lineharbours a provints. The provirus has been crippled so that although itmay produce all the proteins required to assemble infectious viruses,its own RNAA cannot be packaged into virus. RNA produced from therecombinant virus is packaged instead. Therefore, the virus stockreleased from the packaging cells contains only recombinant virus.Non-limiting examples of retroviral packaging lines include PA12, PA317,PE501, PG13, PSLCRIP, RDI 14, GP7C-tTA-G10, ProPak-A (PPA-6), and PT67.Reference is made to Miller fet al., 1986; Miller et al.,

1989; Danos et al., 1988; Pear et al., 1993; and Finer et al., 1994.

Other suitable vectors include adenoviral vectors (see, Frey et al.,1998; and WO 95/27071) and adeno-associated viral vectors. These vectorsare all well known in the art, e.g., as described in Chatterjee et al.,1996; and Stem Cell Biology and Gene Therapy, eds. Quesenbeπy et al.,John Wiley & Sons, 1998; and U.S. Pat. Nos. 5,693,531 and 5,691,176.Theuseof adenovirus-derived vectors may be advantageous under certainsituation because they are not capable of infecting non-dividing cells.Unlike retroviral DNA, the adenoviral DNA is not integrated into thegenome of the target cell. Further, the capacity to carry foreign DNA ismuch larger in adenoviral vectors than retroviral vectors. Theadeno-associated viral vectors are another useful delivery system. TheDNA of this virus may be integrated into non-dividing cells, and anumber of polynucleotides have been successful introduced into differentcell types using adeno-associated viral vectors.

In some embodiments, the construct or vector will include two or moreheterologous polynucleotide sequences. Preferably the additional nucleicacid sequence is a polynucleotide which encodes a selective marker, astructural gene, a therapeutic gene, or a cytokine/chemokine gene.

A selective marker may be included in the construct or vector for thepurposes of monitoring successful genetic modification and for selectionof cells into which DNA has been integrated. Non-limiting examplesinclude drug resistance markers, such as G148 or hygromycin.Additionally negative selection may be used, for example wherein themarker is the HSV-tk gene. This gene will make the cells sensitive toagents such as acyclovir and gancyclovir. The NeoR (neomycin/G 148resistance) gene is commonly used but any convenient marker gene may beused whose gene sequences are not already present in the target cell canbe used. Further non-limiting examples include low-affinity Nerve GrowthFactor (NGFR), enhanced fluorescent green protein (EFGP), dihydrofolatereductase gene (DHFR) the bacterial MsD gene, murine CD24 (HSA), murineCDSa(IyQ, bacterial genes which confer resistance to puromycin orpMeomycin. and β-glactosidaεe.

The additional polynucleotide sequence(s) may be introduced into thehost cell on the same vector or may be introduced into the host cells ona second vector. In a preferred embodiment, a selective marker will beincluded on the same vector as the polynucleotide.

The present invention also encompasses genetically modifying thepromoter region of an endogenous gene such that expression of theendogenous gene is up-regulated resulting in the increased production ofthe encoded protein compared to a wild type adult multipotential cells,

Administration of Stimulatory Factors

Methods of the present invention may involve the use one or morestimulatory factors. Furthermore, compositions of the invention maycomprise one or more stimulatory factors.

In one embodiment, a method of the invention may involve administeringone or more stimulatory factors such as 1α,25-dihydroxyvitamk D₃(1,25D), platelet derived growth factor (PDGF), tumor necrosis factor α(TNF-α), interleukin-1β (IL-1β) and stromal derived factor 1α (SDF-1α)topically, systematically, or locally such as within an implant ordevice.

In particular embodiments, a preferred range for stimulatory factors maybe 0.1 nM-0.1 M, 0.1 nM-0.05 M, 0.05 nM-15 μM or 0.01 uM-10 μM. It is tobe noted that dosage values may vary with the severity of the conditionto be alleviated. For any particular subject, specific dosage regimensmay be adjusted over time according to the individual need and theprofessional judgement of the person administering or supervising theadministration of the compositions. Dosage ranges set forth herein areexemplary only and do not limit the dosage ranges that may be selectedby medical practitioners.

The amount of stimulatory factor in the composition may vary accordingto factors such as the disease state, age, sex, and weight of theindividual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It may be advantageous to formulateparenteral compositions in dosage unit form for case of administrationand uniformity of dosage. “Dosage unit form” as used herein refers tophysically discrete units suited as unitary dosages for subjects to betreated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

It will be appreciated that the stimulatory factor may be administeredin the form of a composition comprising a pharmaceutically acceptablecarrier or excipient.

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. In one embodiment, the carrieris suitable for parenteral administration. Alternatively, the carriercan be suitable for intravenous, intraperitoneal, intramuscular,sublingual or oral administration. Pharmaceutically acceptable carriersinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known, in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Pharmaceutical formulations for parenteral administration may includeliposomes. Liposomes and emulsions are well known examples of deliveryvehicles or carriers that are especially useful for hydrophobic drugs.Depending on biological stability of the therapeutic reagent, additionalstrategies for protein stabilization may be employed. Furthermore, onemay administer the drug in a targeted drug delivery system, for example,in a liposome coated with target-specific antibody. The liposomes willbind to the target protein and be taken up selectively by the cellexpressing the target protein.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, inicroemulsion, liposome, or other orderedstructure suitable to high drug concentration. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol propylene glycol, and liquid polyethyleneglycol, and fee like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.

Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, monostearate salts and gelatin. Moreover, the stimulatoryfactor may be administered in a time release formulation, for example ina composition which includes a slow release polymer. The activecompounds can be prepared with carriers that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations arepatented or generally known to those skilled in the art.

Additionally, suspensions of stimulatory factors may be prepared asappropriate oily suspensions for injection. Suitable lipophilic solventsor vehicles include fatty oils such as sesame oil; or synthetic fattyacid esters, such as ethyl oleate or triglycerides; or liposomes,Suspensions to be used for injection may also contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe compounds to allow for the preparation of highly concentratedsolutions.

Sterile injectable solutions caαbe prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. Ia the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. La accordance with an alternativeaspect of the invention, the stimulatory factor may be formulated withone or more additional compounds that enhance its solubility.

If the compositions are to be administered by inhalation, they may beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser; together with the use of a suitablepropellant, e.g., dichlorodifluoroinethane, tr chlorof uoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof gelatin, for example, for use in an inhaler may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas starch or lactose.

Cellular Compositions

The cellular compositions of the present invention, such as thosecomprising adult multipotential cells, are useful for the regenerationof tissue of various types, including bone, cartilage, tendon, ligament,muscle, skin, and other connective tissue, as well as nerve, cardiac,liver, lung, kidney, pancreas, brain, and other organ tissues.

In some embodiments, the compositions of the present invention may beadministered in combination with an appropriate matrix, for instance,for supporting the cells and providing, a surface for bone, cartilage,muscle, nerve, epidermis and/or other connective tissue growth. Thematrix may be in the form of traditional matrix biomaterials. The matrixmay provide slow release of cells and/or the appropriate environment forpresentation thereof. In some embodiments, various collagenous andnon-collagenous proteins are expected to be upregulated and secretedfrom the cells. This phenomenon accelerates tissue regeneration byenhancing matrix deposition. Matrix proteins can also be expressed inthe genetically engineered cells and enhance the engraftment andattachment of transplanted cells into the transplant area.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the cellular basedcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydxoxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are nonbiodegradable and chemicallydefined, such as sintered hydroxyapatite, bioglass, aluminates, or otherceramics. Matrices may be comprised of combinations of any of the abovementioned types of material, such as polylactic acid and hydroxyapatiteor collagen and tricalcium phosphate. The bioceramics may be altered incomposition, such as in calcivun-aluminate-pliospnate and processing toalter pore size, particle size, particle shape, and biodegradability.

The cellular compositions of the invention may be used to treat patientsrequiring the repair or replacement of cartilage or bone tissueresulting from disease or trauma or failure of the tissue to developnormally, or to provide a cosmetic function, such as to augment facialor other features of the body. Treatment may entail the use of the cellsof the invention to produce new cartilage tissue or bone tissue. Forexample, compositions comprising undifferentiated or chondrogenicdifferentiation-mduced precursor cells may be used to treat a cartilagecondition, for example, rheumatoid arthritis or osteoarthritis or atraumatic or surgical injury to cartilage. As another example,compositions comprising bone precursor cells may be used to treat boneconditions, including metabolic and non-metabolic bone diseases.Examples of bone conditions include meniscal tears, spinal fusion,spinal disc removal, spinal reconstruction, bone fractures, bone/spinaldeformation, osteosarcoma, myeloma, bone dysplasia, scoliosis,osteoporosis, periodontal disease, dental bone loss, osteomalacia,rickets, fibrous osteitis, renal bone dystrophy, and Paget's disease ofbone.

The cellular compositions of the invention may be administered alone oras admixtures with other cells. Cells that may be administered inconjunction with the compositions of the present invention include, butare not limited to, other multipotent or pluripotent cells orchondrocytes, chondroblasts, osteocytes, osteoblasts, osteoclasts, bonelining cells, stem cells, or bone marrow cells. The cells of differenttypes may be admixed with a composition of the invention immediately orshortly prior to administration, or they may be co-cultured together fora period of time prior to administration.

The cellular compositions of the invention may be administered withother beneficial drugs or biological molecules (growth factors, trophicfactors). When the adult multipotential cells are administered withother agents, they may be administered together in a singlepharmaceutical composition, or in separate pharmaceutical compositions,simultaneously or sequentially with the other agents (either before orafter administration of the other agents). Bioactive factors which maybe co-administered include anti-apoptotic agents (e.g., EPO, EPOmimetibody, TPO₅ IGF-I and IGF-II, HGF, caspase inhibitors);anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-betainhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRAMLAST,REMICADE, SIROLIMUS, and NSAIDs (non-steroidal anti-inflammatory drugs;e.g., TEPOXALIN-TOLMETIN, SUPROFEN); immunosupressive/immunomodulatoryagents (e.g., calcineurin inhibitors, such as cyclosporins tacrolimus;mTOR inhibitors (e.g., SIROLIMUS, EVEROLIMUS); anti-proliferativesazathioprine, mycophenolate mofetil); corticosteroids (e.g.,prednisolone, hydrocortisone); antibodies such as monoclonalanti-IL-2Ralpha receptor antibodies (e.g., basiliximab, daclizumab),polyclonal anti-T-cell antibodies (e.g., anti-thymocyte globulin (ATG);anti-lymphocyte globulin (ALG); monoclonal anti-T cell antibody OKT3));anti-thrombogenio agents (e.g., heparin, heparin derivatives, urokinase,PPack (dextrophenylalanine proline arginine chloromethylketone),antithrombin compounds, platelet receptor antagonists, anti-thrombinantibodies, anti-platelet receptor antibodies, aspirin, dipyridamole,protamine, hirudin, prostaglandin inhibitors, and platelet inhibitors);and anti-oxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol,coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine) as well aslocal anesthetics. As another example, the cells may be co-administeredwith scar inhibitory factor as described in U.S. Pat. No. 5,827,735,incorporated herein by reference.

In one embodiment, cellular compositions of the invention areadministered as undifferentiated cells, i.e., as cultured in GrowthMedium. Alternatively, the cellular compositions way be administeredfollowing exposure in culture to conditions that stimulatedifferentiation toward a desired phenotype, for example, an osteogenicphenotype.

The cellular compositions of the invention may be surgically implanted,injected, delivered (e.g., by way of a catheter or syringe), orotherwise administered directly or indirectly to the site in need ofrepair or augmentation. The cells may be administered by way of a matrix(e.g., a three-dimensional scaffold). The cells may be administered withconventional pharmaceutically acceptable carciers. Routes ofadministration of the cells of the invention or compositions orcomponents (e.g., ECM, cell lysate, conditioned medium) thereof includeintramuscular, ophthalmic, parenteral (including intravenous),intraarterial, subcutaneous, oral, and nasal administration. Particularroutes of parenteral administration include, but are not limited to,intramuscular, subcutaneous, intraperitoneal, intracerebral,intraventricular, intracerebroventricular, intrathecal, intracistemal,intraspinal and/or peri-spinal routes of administration.

When cells are administered in semi-solid or solid devices, surgicalimplantation into a precise location in the body is typically a suitablemeans of administration. Liquid or fluid pharmaceutical compositions,however, may be administered to a more general location (e.g.,throughout a diffusely affected area, for example), from which theymigrate to a particular location, e.g., by responding to chemicalsignals.

Other embodiments encompass methods of treatment by administeringpharmaceutical compositions comprising cellular components (elg., celllysates or components thereof) or products (e.g., extracellular matrix,trophic and other biological factors produced through geneticmodification).

Dosage forms and regimes for administering cellular compositionsdescribed herein are developed in accordance with good medical practice,taking into account the condition of the individual patient, e.g.,nature and extent of the condition being treated, age, sex, body weightand general medical condition, and other factors known to medicalpractitioners. Thus, the effective amount of a pharmaceuticalcomposition to be administered to a patient is determined by theseconsiderations as known in the art.

In some embodiments of the invention, it may not be necessary ordesirable to immunosuppress a patient prior to initiation of therapywith cellular compositions of the present invention. Accordingly,transplantation with allogeneic, or even xenogeneic, adultmultipotential cells may be tolerated in some instances.

However, in other instances it may be desirable or appropriate topharmacologically immunosuppress a patient prior to initiating celltherapy. This may be accomplished through the use of systemic or localimmunosuppressive agents, or it may be accomplished by delivering thecells in an encapsulated device. Adult multipotential cells may beencapsulated in a capsule that is permeable to nutrients and oxygenrequired by the cell and therapeutic factors the cell is yet impermeableto immune humoral factors and cells. Preferably the encapsulant ishypoallergenic, is easily and stably situated in a target tissue, andprovides added protection to the implanted structure. These and othermeans for reducing or eliminating an immune response to the transplantedcells are known in the art, As an alternative, adult multipotential.cells may be genetically modified to reduce their immunogenicity.

Survival of transplanted cells in a living patient can be determinedthrough the use of a variety of scanning techniques, e.g., computerizedaxial tomography (CAT or CT) scan, magnetic resonance imaging (MRI) orpositron emission tomography (PET) scans. Determination of transplantsurvival can also be done post mortem by removing the target tissue, andexamining it visually or through a microscope. Alternatively, cells canbe treated with stains that are specific for cells of a specificlineage. Transplanted cells can also be identified by priorincorporation of tracer dyes such as rhodamine-oτ fluorescein-labeledmicrospheres, fast blue, bisbenzamide, ferric microparticles, orgenetically introduced reporter gene products, such asbeta-galactosidase or beta-glucuronidase.

Functional integration of transplanted cells into a subject can beassessed by examining restoration of the function that was damaged ordiseased, for example, restoration of joint or bone function, oraugmentation of function.

Cellular compositions of the invention may include one or more bioactivefactors, for example but not limited to a growth factor, adifferentiation-inducing factor, a cell survival factor such as caspaseinhibitor, or an anti-inflammatory agent such as p38 kinase inhibitor.

Alternatively, cells to be transplanted may be genetically engineered toexpress such growth factors, antioxidants, antiapoptotic agents, oranti-inflammatory agents.

Pharmaceutical compositions of the invention may comprise homogeneous orheterogeneous populations of cells, extracellular matrix or cell lysatethereof, or conditioned medium thereof in a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers for the cells of theinvention include organic or inorganic carrier substances suitable whichdo not deleteriously react with the cells of the invention orcompositions or components thereof. To the extent they arebiocompatible, suitable pharmaceutically acceptable carriers includewater, salt solution (such as Ringer's solution), alcohols, oils,gelatins, and carbohydrates, such as lactose, amylose, or starch, fattyacid esters, hydroxymethylcellulose, and polyvinyl pyro-lidine. Suchpreparations can be sterilized, and if desired, mixed with auxiliaryagents such as lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, andcoloring, Pharmaceutical carriers suitable for use in the presentinvention are known in the art and are described, for example, inPharmaceutical Sciences (17th Ed., Mack Pub. Co., Eastøn, Pa.) and WO96/05309.

One or more other components may be added to transplanted cells,including selected extracellular matrix components, such as one or moretypes of collagen known in the art, and/or growth factors, platelet-richplasma, and drugs. Alternatively, the cells of the invention may begenetically engineered to express and produce for growth factors.Details on genetic engineering of the cells of the invention areprovided herein.

IQ a non-limiting embodiment, a formulation comprising the cells of theinvention is prepared for administration directly to the site where theproduction of new tissue, such as bone tissue, is desired. For example,and not by way of limitation, the cells may be suspended in a hydrogelsolution for injection. Examples of suitable hydrogels for use in theinvention include self-assembling peptides, such as RAD1 6.Alternatively, the hydrogel solution containing the cells may be allowedto harden, for instance in a mold, to form a matrix having cellsdispersed therein prior to implantation. Or, once the matrix hashardened, the cell formations may be cultured so that the cells aremitotically expanded prior to implantation. The hydrogel is an organicpolymer (natural or synthetic) which is cross-linked via covalent,ionic, or hydrogen bonds to create a three-dimensional open-latticestructure which entraps water molecules to form a gel. Examples ofmaterials which can be used to form a hydrogel include polysaccharidessuch as alginate and salts thereof, peptides, polyphosphazines, andpolyacrylates, which are cross-linked ionically, or block polymers suchas polyethylene oxide-polypropylene glycol block copolymers which arecrosslinked by temperature or pH, respectively. In some embodiments, thesupport for the cells is biodegradable.

In some embodiments of the invention, the formulation comprises an insitu polyrnemable gel, as described, for example, in U.S. PatentApplication Publication 2002/0022676; Anseth et al, 2002; and Wang etal., 2003.

In some embodiments, the polymers are at least partially soluble inaqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers ox polymers canalso be used. Examples of acidic groups axe carboxylic acid groups,sulfonic acid groups, halogepated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

Examples of polymers with basic side groups that can be reacted withanions are ρoly(vinyl amines), poly(vinyl pyridine), polyvinylimidazole), and some immo substituted polyphosphates. The ammonium orquaternary salt of the polymers can also be formed from the backbonenitrogens or pendant imino groups. Examples of basic side groups areamino and imiπo groups.

Alginate can be ionically cross-linked with divalent cations, in water,at room temperature, to form a hydrogel matrix. Due to these mildconditions, alginate has been the most commonly used polymer forhybridoma cell encapsulation, as described, for example, in U.S. Pat.No. 4,352,883 to Lim. In the Lim process, an aqueous solution containingthe biological materials to be encapsulated is suspended in a solutionof a water soluble polymer, the suspension is formed into droplets whichare configured into discrete microcapsules by contact with multivalentcations, then the surface of the microcapsules is crosslinked withpolyamino acids to form a semipermeable membrane around the encapsulatedmaterials.

Polyphosphazenes are polymers with backbones consisting of nitrogen andphosphorous separated by alternating single and double bonds. Eachphosphorous atom is covalently bonded to two side chains.

The polyphosphazenes suitable for cross-linking have a majority of sidechain groups which are acidic and capable of forming salt bridges withdi- or trivalent cations. Examples of preferred acidic side groups arecarboxylic acid groups and sulfonic acid groups. Hydrolytically stablepolyphosphazenes are formed of monomers having carboxylic acid sidegroups that are crosslinked by divalent or trivalent cations such asCa²⁺ or Al³⁺. Polymers can be synthesized that degrade by hydrolysis byincorporating monomers having imidazole, amino acid ester, or glycerolside groups. For example, a polyanionicpoly[bis(carboxylatophealoxy)]phosphazene (PCPP) can be synthesized,which is cross-linked with dissolved multivalent cations in aqueousmedia at room temperature or below to form bydr αgel matrices.

Biodegradable polyphosphazenes have at least two differing types of sidechains, acidic side groups capable of forming salt bridges withmultivalent cations, and side groups that hydrolyze under-in vivoconditions, e.g., imidazole groups, amino acid esters, glycerol andglucosyl.

Hydrolysis of the side chain results in erosion of the polymer. Examplesof hydrolyεing side chains are unsubstituted and substituted imidazolesand amino acid esters in which the group is bonded to the phosphorousatom through an amino linkage (polyphosphazene polymers in which both Rgroups are attached in this manner are known as polyaminophosphazenes).For polyimidazolephosphazenes, some of the “R” groups on thepolyphosphazene backbone are imidazole rings, attached to phosphorous inthe backbone through a ring nitrogen atom, Other “R” groups can beorganic residues that do not participate in hydrolysis, such as methylphenoxy groups or other groups shown in the scientific paper of Allcocket al. (1977). Methods of synthesis of the hydrogel materials, as wellas methods for preparing such hydrogels, are known in the art.

Other components may also be included in the formulation, including butnot limited to any of the following: (1) buffers to provide appropriatepH and isotonicity; (2) lubricants; (3) viscous materials to retain thecells at or near the site of administration, including, for example,alginates, agars and plant gums; and (4) other cell types that mayproduce a desired effect at the site of administration, such as, forexample, enhancement or modification of the formation, of tissue or itsphysicochemical characteristics, or as support for the viability of thecells, or inhibition of inflammation or rejection. The cells may becovered by an appropriate wound covering to prevent cells from leavingthe site. Such wound coverings are known as those of skill in the art.

Fibrin Glue

Fibrin glues are a class of surgical sealants which have been used invarious clinical settings. As the skilled address, would be aware,numerous sealants are useful in compositions of the invention. However,a preferred embodiment of the invention relates to the use of fibringlues with cells of the invention.

When used herein the term “fibrin glue” refers to the insoluble matrixformed by the cross-linking of fibrin polymers in the presence ofcalcium ions. The fibrin glue may be framed from fibrinogen, or aderivative or metabolite thereof, fibr n (soluble monomers or polymers)and/or complexes thereof derived from biological tissue or fluid which,forms a fibrin matrix. Alternatively, the fibrin glue may be formed fromfibrinogen, or a derivative or metabolite thereof, or fibrin, producedby recombinant DNA technology.

The fibrin glue may also be formed by the interaction of fibrinogen anda catalyst of fibrin glue formation (such as thrombin and/or FactorXIII). As will be appreciated by those sMlled in the art, fibrinogen isproteolytically cleaved in the presence of a catalyst (such as thrombin)and converted to a fibrin monomer. The fibrin monomers may then formpolymers which may cross-link to form a fibrin glue matrix. Thecross-linking of fibrin polymers may be enhanced by the presence of acatalyst such as Factor Xiπ. The catalyst of fibrin glue formation maybe derived from blood plasma, cryoprecipitate or other plasma fractionscontaining fibrinogen or thrombin. Alternatively, the catalyst may beproduced by recombinant DNA technology.

The rate at which the clot forms is dependent upon the concentration ofthrombin mixed with fibrinogen. Being an enzyme dependent reaction, thehigher the temperature (up to 37° C.) the faster the clot formationrate. The tensile strength of the clot is dependent upon theconcentration of fibrinogen used.

Use of fibrin glue and methods for its preparation and use are describedby I-firεh et al. in U.S. Pat. No. 5,643,192. Hirsh discloses theextraction of fibrinogen and thrombin components from a single donor,and the combination of only these components for use as a fibrin glue.Marx, U.S. Pat. No. 5,651,982, describes another preparation and methodof use For fibrin glue. Marx provides a fibrin glue with liposomes foruse as a topical sealant in mammals. The preparation and use of atopical fibrinogen complex (TFC) for wound healing is known in thefield. PCT. Application No. PCT/US95/15576, PCT Publication No,WO96/17633—of The American Red Cross discusses TFC preparationscontinuing fibrinogen, thrombin, and calcium chloride, for example, atpages 16-18 of PCT Publication No. WO96/17633.

Several publications describe the use of fibrin glue for the delivery oftherapeutic agents. For example, U.S. Pat. No. 4,983,393 discloses acomposition for use as an intra-vaginal insert comprising agarose, agar,saline solution glycosaminoglycans, collagen, fibrin and an enzyme.Further, U.S. Pat. No. 3,089,815 discloses an injectable pharmaceuticalpreparation composed of fibrinogen and thrombin and U.S. Pat. No.6,468,527 discloses a fibrin glue which facilitates the delivery ofvarious biological and non-biological agents to specific sites withinthe body.

Formulation of a Bone Tissue Patch

Culture or co-cultures of cells of the invention in a pre-shaped wellenables the manufacture of a tissue patch of pre-determined thicknessand volume. The volume of the resulting tissue patch is dependent uponthe volume of the well and upon the number of adult multipotential cellsin the well. Tissue of optimal pre-determined volume may be prepared byroutine experimentation by altering either or both of the aforementionedparameters.

The cell contacting surface of the well may be coated with a moleculethat discourages adhesion of adult multipotential cells to the cellcontacting surface. Preferred coating reagents include silicon basedreagents i.e., dichlorodimethylsilane or polytetrafluoroethylene basedreagents, i.e., TEFLON. Procedures for coating materials with siliconbased reagents, specifically dichlorodimethylsilane, are well known inthe art. See for example, Sambrook et al. (1989). “Molecular Cloning ALaboratory Manual”. Cold Spring Harbor Laboratory Press, the disclosureof which is incorporated by reference herein. It is appreciated thatother biocompatible reagents that prevent the attachment of cells to thesurface of the well may be useful in the practice of the instantinvention.

Alternatively, the well may be cast from a pliable or moldablebiocompatible material that does not permit attachment of cells per se.Preferred materials that prevent such cell attachment include, but arenot limited to, agarose, glass, untreated cell culture plastic andpolytetrafluoroethylene, i.e., TEFLON, Untreated cell culture plastics,i.e., plastics that have not been treated with or made from materialsthat have an electrostatic charge are commercially available, and may bepurchased, for example, from Falcon Labware, Becton-Dickinson, LincolnPark, N.J. The aforementioned materials, however, are not meant to belimiting. It is appreciated that any other pliable or moldablebiocompatible material that inherently discourages the attachment ofadult multipotential cells may be useful in the practice of the instantinvention.

Cells of the invention in suspension may be seeded into and cultured inthe pre-shaped well. The cells may be induced to differentiate to achondrogenic or osteogenic phenotype in culture in the well or may havebeen induced to differentiate prior to seeding in the well. The cellsmay be diluted by the addition of culture medium to a cell density ofabout 1×10⁵ to 1×10⁹ cells per milliliter.

The cells may form a cohesive plug of cells. The cohesive plug of cellsmay be removed from the well and surgically implanted into the tissuedefect. It is anticipated that any undifferentiated cells, such as adultmulitpotential cells of invention, may differentiate in situ thereby toform tissue in vivo.

Bone defects may be identified inferentially by using computer aidedtomography (CAT scanning); X-ray examination, magnetic resonance imaging(MRI), analysis of synovial fluid or serum markers or by any otherprocedures known in the art. Defects in mammals also are readilyidentifiable visually during arthroscopic examination or during opensurgery of the joint. Treatment of the defects can be effected during anarthroscopic or open surgical procedure using the methods andcompositions disclosed herein.

Accordingly, once the defect has been identified, the defect may betreated by the following steps of (1) surgically implanting at thepre-determined site a tissue patch prepared by the methodologiesdescribed herein, and (2) permitting the tissue patch to integrate intopre-determined site.

The tissue patch optimally has a size and shape such that when the patchis implanted into the defect, the edges of the implanted tissue contactdirectly the edges of the defect. In addition, the tissue patch may befixed in place during the surgical procedure. This can be effected bysurgically fixing the patch into the defect with biodegradable suturesand/or by applying a bioadhesive to the region interfacing the patch andthe defect.

In some instances, damaged tissue may be surgically excised prior to theimplantation of the patch of tissue.

Transplantation of Cells Usinff Scaffolds

The cellular compositions of the invention or co-cultures thereof may beseeded onto or into a three-dimensional scaffold and implanted in vivo,where the seeded cells will proliferate on the framework and form areplacement tissue, such as bone tissue in vivo in cooperation with thecells of the patient.

For example, but not by way of limitation, the scaffold may be designedsuch that the scaffold structure: (1) supports the seeded cells withoutsubsequent degradation; (2) supports the cells from the time of seedinguntil the tissue transplant is remodeled by the host tissue; (2) allowsthe seeded cells to attach, proliferate, and develop into a tissuestructure having sufficient mechanical integrity to support itself invitro, at which point, the scaffold is degraded. A review of scaffolddesign is provided by Hutmacher (2001).

Scaffolds of the invention can be administered in combination with anyone or more growth factors, cells, for example stem cells, bone marrowcells, chondrocytes, chondroblasts, osteocytes, osteoblasts,osteoclasts, bone lining cells, or their precursors, drugs or othercomponents described above that stimulate tissue formation or otherwiseenhance or improve the practice of the invention. The cells of theinvention to be seeded onto the scaffolds may be genetically engineeredto express growth factors or drugs.

The cells of the invention can be used to produce new tissue in vitro,which can then be implanted, transplanted or otherwise inserted into asite requiring tissue repair, replacement or augmentation in a patient

In a non-limiting embodiment, the cells of the invention are used toproduce a three-dimensional tissue construct in vitro, which is thenimplanted in vivo. As an example of the production of three-dimensionaltissue constructs, see U.S. Pat. No. 4,963,489, which is incorporatedherein by reference. For example, the cells of the invention may beinoculated or “seeded” onto a three-dimensional framework or scaffold,and proliferated or grown in vitro to form a living tissue that can beimplanted in vivo.

The cells of the invention can be grown freely in a culture vessel tosub-confmβncy or confluency, lifted from the culture and inoculated ontoa three-dimensional framework.

Inoculation, of the three-dimensional framework with a highconcentration of cells, e.g., approximately 10⁶ to 5×10⁷ cells permilliliter, will result in the establishment of the three-dimensionalsupport in relatively shorter periods of time.

Examples of scaffolds which may be used in the present invention includenonwoven mats, porous foams, or self assembling peptides. Nonwoven matsmay, for example, be formed using fibers comprised of a syntheticabsorbable copolymer of glycolic and lactic acids (PGA/PLA), sold underthe tradename VICRYL (Ethicon, Inc., Somerville, N.J.). Foams, composedof, for example, poly(epsilon-caprolactone)/poly(glycolic acid)(PCL/PGA) copolymer, formed by processes such as freeze-drying, orlyophilized—as discussed in U.S. Pat, No. 6,355,699, are also passablescaffolds. Hydrogels such as self-assembling peptides (e.g., RAD16) mayalso be used. These materials are frequently used as supports for growthof tissue.

The three-dimensional framework may be made of ceramic materialsincluding, but not limited to: mono-, di-, tri-, alpha-tri-, beta-tri-,and tetra-calcium phosphate, hydroxyapatite, fluoroapatites, calciumsulfates, calcium fluorides, calcium oxides, calcium carbonates,magnesium calcium phosphates, biologically active glasses such asBIOGLASS (University of Florida, Gainesville, Fla.), and mixturesthereof. There are a number of suitable porous biocompatible ceramicmaterials currently available on the commercial market such as SURGIBON(Unilab Surgibone, Inc., Canada), ENDOBON (Merck Biomaterial France,France), CEROS (Mathys, A-G., Bettlach, Switzerland), and INTERPORE(Interpoie. Irvine, Calif., United States), and mineralized collagenbone grafting products such as HEALOS (Orquest, Inc., Mountain View,Calif.) and VITOSS, RHAKOSS- and CORTOSS (Orthovila, Malvern, Pa.). Theframework may be a mixture, blend or composite of natural and/orsynthetic materials. In some embodiments, the scaffold is in the form ofa cage. In a preferred embodiment, the scaffold is coated with collagen.

According to a preferred embodiment, the framework is a felt, which canbe composed of a multifilament yarn made from a bioabsorbable material,e.g., PGA, PLA, PCL copolymers or blends, or hyaluronic acid. The yarnis made into a felt using standard textile processing techniquesconsisting of crimping, cutting, carding and needling.

In another preferred embodiment the cells of the invention are seededonto foam scaffolds that may be composite structures. In addition, thethree-dimensional framework may be molded into a useful shape, such asthat of the external portion of the ear, a bone, joint or other specificstructure in the body to be repaired, replaced or augmented.

In another preferred embodiment, the cells are seeded onto a frameworkcomprising a prosthetic device for implantation iato a patient, asdescribed in U.S. Pat. No. 6,200,606, incorporated herein by reference.As described therein, a variety of clinically useful prosthetic deviceshave been developed for use in bone and cartilage grafting procedures,(see e.g. Bone Grafts and Bone Substitutions. Ed. M. B. Habal & A. H.Reddi, W. B. Saunders Co., 1992). For example, effective knee and hipreplacement devices have been and continue to be widely used in theclinical environment Many of these devices are fabricated using avariety of inorganic materials having low immunogenic activity, whichsafely function in the body. Examples of synthetic materials which havebeen tried and proven include titanium alloys, calcium phosphate,ceramic hydroxyapatite, and a variety of stainless steel andcobalt-chrome alloys. These materials provide structural support and canform a scaffolding into which host vascularization and cell migrationcan occur.

The framework may be treated prior to inoculation of the cells of theinvention in order to enhance cell attachment. For example, prior toinoculation with the cells of the invention, nylon matrices could betreated with 0.1 molar acetic acid and incubated in polyiysine, PBS,and/or collagen to coat the nylon. Polystyrene could be similarlytreated using sulfuric acid.

In addition, the external surfaces of the three-dimensional frameworkmay be modified to improve the attachment or growth of cells anddifferentiation of tissue, such as by plasma coating the framework oraddition of one or more proteins (e.g., collagens, elastic fibers,reticular fibers), glycoproteins, glycosamhioglycans (e.g., heparinsulfate, chondroitin-4-sulfate, chondroitin.-6-sulfate, dermatansulfate, keratin sulfate), a cellular matrix, and/or other materialssuch as, but not limited to, gelatin, alginates, agar, agarose, andplant gums, among others.

In some embodiments, the scaffold is comprised of or is treated withmaterials that render it non-thrombogenic. These treatments andmaterials may also promote and sustain endothelial growth, migration,and extracellular matrix deposition. Examples of these materials andtreatments include but are not limited to natural materials such asbasement membrane proteins such as laminin and Type IV collagen,synthetic materials such as ePTFE, and segmented polyurethaneureasilicones, such as PURPANN (The Polymer Technology Group, Inc.,Berkeley, Calif.). These materials can be further treated to render thescaffold non-thrombogenic. Such treatments include anti-thromboticagents such as heparin, and treatments which alter the surface charge ofthe material such as plasma coating.

In some embodiments, the surface of the scaffold is textured. Forexample, in some aspects of the invention, the scaffold is provided witha groove and ridge pattern. The grooves ate preferably less than about500 microns, more preferably less than about 100 microns, and mostpreferably between about 10 nanometers and 10 microns, Such“microgrooves” allow the cells to align and/or migrate guided by thesurface grooves.

In some embodiments, it is important to re-create in culture thecellular microenvironment found in vivo, such that the extent to which,the cells of the invention are grown prior to implantation in vivo oruse in vitro may vary. In addition, growth factors, ohondrogenicdifferentiation inducing agents, osteogenic inducing agents, andangiogenic factors may be added to the culture medium prior to, during,or subsequent to inoculation of the cells to trigger differentiation andtissue formation by the cells or progeny thereof.

The three-dimensional framework may be modified so that the growth ofcells and the production of tissue thereon is enhanced, or so that therisk of rejection of the implant is reduced. Thus, one or morebiologically active compounds, including, but not limited to,antiinflammatories, immunosuppressants or growth factors, may be addedto the framework.

EXAMPLES

The present invention will now be described in more detail withreference to the following non-limiting examples.

Example 1 Generation of the mAb STRO-3 Materials and Methods Human MPCCultures

Stromal cultures were established, essentially as described previously(Gronthos et al., 2003). The use of normal bone marrow (BM) cells forthese studies was approved by the Human Ethics Committee of the RoyalAdelaide Hospital, Australia. After washing thrice in “HHF” (HanksBuffered Salt Solution (HBSS) supplemented with 2 OmM HEPES and 5% FCS),the 1-5×10⁷ bone marrow mononuclear cells (BMMNCs) were resuspended in:10 ml of alpha modification of Eagles' medium (α-MEM: Flow Laboratories,Irvine, Scotland) supplemented with Folic acid (0.01 mg/ml),myo-inositol (0.4 mg/ml) (Sigma Chemical Co. St Louis, Mo.), 50 mM/L2-mercaρtoethanol, imM/L hydrocortisone sodium succinate (Sigma), 12.5%FCS, and 12.5% horse serum (CSL, Melbourne, Australia) and cultured in25 cm² flasks (Becton Dickinson Labware, Franklin Lakes, N.J.), Upondevelopment of a confluent stromal layer, the cells were detached using0.05% (w/v) trypsin-EDTA in PBS (Gibco) and replated in the same mediumat approximately 1-2×10⁴ cells per cm² in 2×75 cm² tissue culture flasks(Becton Dickinson Labware, Franklin Lakes, N.J.).

Results and Discussion

A panel of mouse monoclonal antibodies reactive with human MPC weregenerated following the fusion of splenocytes derived from miceimmunized with cultured human BM stromal cells. Preliminary screens weredesigned to identify mAbs which reacted with normal human bone cells(NHBC) and MPC but exhibited limited reactivity with the majority ofBMMNC. One mAb, STRO3, was initially selected for further analysis dueto its unique pattern of reactivity with different cell lines. Tertiaryclones of the STRO-3 hybridoma were isolated by limiting dilution in96-well plates and subsequently screened as described above. Thedistribution patterns of STRO-3 with various stromal cell types aresummarised in Table 1. The mAb STRO-3 exhibited reactivity to aproportion of NHBC and MPC and with only a minor proportion of BMMNC.The immunoglobulin isotype of STRO-3 from tertiary hybridomasupernatants was determined to be IgGi using an isotype detection kit(Roche).

TABLE 1 Immunoreactivity of STRO-3 supernatants from tertiary clonedhybridomas on different cell types using in situ immunofluorecence asdescribed in the methods. In Situ Immunofluoresce Cell Type StainingPeripheral blood mononuclear cells NS Bone marrow mononuclear cells +/−(neutophils) Ex vivo expanded MPC +/− Cultured normal human bone cells++/− Human foreskin fibroblasts +/− Human ubilical vein endotlielialcells NS Murine bone marrow stromal line BMS2 NS Human osteosarcoma cellline SAOS-2 ++ Human osteosarcoma cell line MG63 NS Human osteosarcomacell line HOS ++ + Low fluorescence staining on all cells ++ Highfluorescence staining on all cells NS No fluorescence staining on cells+/−, -H-/− Fluorescence Staining on a subpopulation of cells

Example 2 Molecular Characterization of the STRO-3 Antigen Materials andMethods

Expression Cloning of the cDNA Encoding STRO-3Antigen

The cDNA encoding the cell surface antigen identified by the mAb STRO-3was isolated from a human bone marrow stromal cell cDNA library hi theretroviral vector, pRUFneo as described (Zannettino et al., 1996).Briefly, cDNA synthesised from mRNA from HBMSC cultures was cloned intothe retroviral vector pRUFneo. Plasmid DNA from the library was used totransfect a viral packaging line (PA317). Virus containing supernatantfrom these cells was used to infect the packing cell line ψ2, which inturn was used to infect the murine factor-dependent cell line BAF-3.Infected cells were selected for G418 resistance, labelled with STRO-3antibody and cells specifically binding the antibody were isolated byimmunomagnetic bead selection (Dynabead, Dynal, Oslo, Sweden). Afterexpansion of the initially selected cells in culture, immunomagneticbead selection was repeated a further two times. BAF-3 cells whichdemonstrated specific binding of STRO-3 antibody (approximately 60%)were purified by florescence-activated cell sorting (FACS) and clonesisolated following culture in semi-solid media as previously (Zannettinoet al., 1996). To recover proviral cDNA inserts corresponding to theSTRO-3 antigen, the polymerase chain reaction (PCR) using retroviralspecific primers was performed on genomic DNA prepared from threeSTRO-3-expressing BAF-3 clones, as previously described (Zannettino etal., 1996).

Partial-Sequencing of PCR-rescuedcDNA Clones and Computer Analysis:

As described previously (Zannettino et al., 1996), cDNA clones generatedby PCR were gel purified and subcloned into the pGEM-T vector (Promega,Madison, Wis.) as recommended by the manufacturer. Double-stranded DNAwas prepared by standard alkaline lysis “mini-prep” method (Qiagenminiprep Kit) and 1-2 μg was used per sequencing reaction. Reactionswere prepared using the PRISM™ Ready Reaction Cycle sequencing kit(Applied Biosystem, Foster City, Calif.), as recommended by themanufacturer. Reactions analysing both cDNA strands were run on aApplied Biosystems 373 automated sequence analyser and 500-600 bp of 5′and 3′ sequence data was routinely obtained per clone. Sequence datawere then analysed by accessing the Genbank and European MolecularBiology laboratory (EMBL) data bases at the National Centre forBiotechnological Information (NCBI).

Recloning of the STRO-3 Antigen cDNA Clone into pRVFne σ and Validationof Surface Antigen Expression

Following PCR recovery of proviral cDNA inserts from genomic DNA, uniqueBam HI and Xho I restriction sites present in the 5′ and 3′ flankingregions respectively, were utilised to “redone” the cDNA into the MCS ofthe retroviral vector pRUFneo. E. coli DHlOB cells were transformed andplasmid DNA isolated using Qiagen-tip 100 columns (Qiagen, Victoria,Australia) as recommended by the manufacturer. Stable, G418 resistant ψ2virus-producing cell lines were produced by calcium phosphatetransfection and used to infect BAF-3 cells by co-cultivation, asdescribed previously (Zannettino et al., 1996), G418 resistant FDC-Plcells were then analysed for antigen expression by indirectimmunofluorescence and flow cytometry.

Reverse Transcriptase Polymerase Chain Reaction (RT˜PCS) Analysis.

Total cellular RNA was prepared from clones of STRO-3 antigen BAP-3cells using RNAzolB extraction method (Biotecx Lab. Inc., Houston,Tex.), according to the manufacturer's recommendations. RNA isolatedfrom each subpopulation was then used as a template for cDNA synthesis,prepared using a First-strand cDNA synthesis kit (Pharmacia Biotech,Uppsala, Sweden). The expression of bone and Jiver/kidney isoform of ALPtranscripts was assessed by PCR amplification, using a standard protocolas described previously (Groathos et al, 1999). The alkaline phosphataseprimer sets used in this study have been previously described (Sato etal., 1994). Following amplification, each reaction mixture was analysedby 1.5% agarose gel electrophoresis, and visualised by ethidiuni bromidestaining. RNA integrity was assessed by the expression of GAPDH(Gronthos et al., 1999),

Results and Discussion

Using a retroviral expression library strategy pioneered in ourlaboratory, we subsequently identified the gene encoding for the proteinidentified by the STRO-3 mAb (Zannettino et al., 1996). Briefly, themurine cell line BAF-3, was infected with retroviral particlesconstructed from a library of cDNAs derived from cultured human MPC.BAF-3 clones reactive with STRO-3 were isolated by immunomagnetic beadselection using sheep anti-mouse IgG magnetic beads.

As shown in FIG. 1, flow cytometric analysis of cells which wererecovered following several rounds of bead selection were found toexpress the STRO-3 antigen at appreciable levels. Clones ofSTRO-3-expressing BAF-3 cells were subsequently prepared by seeding thepool of immunomagnetic bead-selected cells at low density intosemi-solid methylcellulose, as described in the methods. A randomisedselection of BAF-3 colonies were then isolated and expanded in liquidculture supplemented with murine IL-3 and G418-Selected clonesdemonstrating a high reactivity with STRO-3 were then expanded inculture, and genomic DNA prepared as described in the methods. The cDNAinserts were subsequently rescued from the provims by long-range PCRamplification as previously described (Zannettino et al., 1996).

PCR amplification of a representative clone for STRO-3 is shown in FIG.2. Following agarose gel electrophoresis and ethidium bromide staining,the corresponding PCR products from three different clones weregel-puréed using a QIAquick Gel Extraction Kit (QIAGEN Inc. Chatsworth,Calif., USA) and cloned in the pCxEM-T vector as recommended by themanufacturer. The nucleotide sequence of the PCR products was derived bysequencing with the PRISM™ Ready Reaction DyeDeoxy™ Terminator CycleSequencing Kit (Applied Biosystems Inc., Foster City, Calif., USA)according to the manufacturer's specifications. Reactions were run on aApplied Biosystems 373 automated sequence analyser, and several hundredbase pairs of nucleic acid sequence data was obtained for each clone.The resulting partial-nucleotide sequences were compared with theentries submitted to the Genbank/EMBL databases via standard FASTAalignment analysis. Partial DNA sequences for both antigens are given(FIG. 3). Comparisons with sequences in the combined EMBL/Genbankdatabase identified the STRO-3 antigen as corresponding to thebone/liver/kidney form of alkaline phosphatase, namely TNAP.

Independent confirmation of the specificity of the STRO-3 mAb wassubsequently obtained following immunofluorescence and flow cytometricanalysis of TNAP expressing BAF-3 clones with the alkaline phosphatasespecific antibodies, B4-78, 50 (Developmental Studies Hybridoma Bank,University of Iowa) which recognises an epitope conserved between eachof the bone, liver and kidney isoforms of ALP (FIG. 4).

In addition, the mAb B4-50 (Developmental Studies Hybridoma Bank,University of Iowa) which has been previously shown to be specific forthe bone AP enzyme also displayed immunoreactivity with the TNAPtransfectants. In contrast, no detectable reactivity was observedfollowing the staining of the TNAP transfectants with mAb 8B6 ((DAKO),which identifies an epitope present on only human placental AP antigen.In addition, BAF-3 cells re-transfected with the TNAP-BAF-3 cDNA insert,were found to express an active form of alkaline phosphatase, asdemonstrated by positive reactivity in the presence of alkalinephosphatase substrate (FIG. 5). PCR analysis using specific primers forthe bone and liver forms of alkaline phosphatase (Sato et al., 1994)identified the transcripts as bone-specific (FIG. 6).

Example 3 Immunohistochemical Detection of TNAP by STRO-3 mAb inSections of BM Trephine Materials and Methods

Five micron sections of paraffin-embedded normal post-natal bone, werecut onto 3-aminopropyl-triethoxysilane-coated slides and endogenousperoxidase activity blocked by incubation with 3% H₂O₂/Methanol.Microwave antigen retrieval was then performed in the presence of 1mM.EDTA, pH 8.0 buffer. The slides were allowed to cool to 40° C. andnon-specific binding blocked by incubating sections with 3% noπrialhorse serum for 1 hour at RT, The slides were then incubated overnightwith either an isotype-matched, non-binding control mAb (1B5, IgGi) orSTRO-3 mAb. Bound antibody was revealed using a three-stepimmunoperoxidase method (Gronthos et al., 2000; Gronthos et al., 2003)in which slides were sequentially incubated with (a) affinity-purifiedHRP-conjugated goat anti-mouse antibody (Dako, Botany, NSW, Australia),followed by (b) affinity-purified Horseradish peroxidase(HRP)-conjugated swine anti-goat immunoglobulin (Tago, Burlingame,Calif. USA) and (c) hydrogen peroxide as substrate and aminoethylcarbazole (AEC, Sigma, St Louis, Mo.) as the dye. Slides werecounterstained briefly with haematoxylin solution and mounted in GuirAquamount (BDH, Poole, UK).

Results and Discussion

The immunoreactivity of STRO-3 nxAb was assessed in sections ofdeveloping bone marrow derived from 55 day old human limb. No stainingwas observed in the periosteum or in cartilage (FIG. 7). However therewas a marked expression of TNAP in the mesenchymal cells of the bonemarrow spaces, perivascular regions and at the interface of the growthplate region.

Example 4 Isolation of Human Bone Marrow Cells Using STRO-3 mAb

Bone marrow (BM) is harvested from healthy normal adult volunteers(20-35 years old), in accordance with procedures approved by theInstitutional Ethics Committee of the Royal Adelaide Hospital. Briefly,40 ml of BM is aspirated from the posterior iliac crest intolithium-heparin anticoagdantaonlaining tubes. BMMNC are prepared bydensity gradient separation using Lymphoprep™ (Nycomed Pharnra, Oslo,Norway) as previously described (Zannetti

αo et al.₃ 199S). Following centrifugation at 400×g for minutes at 4°C., the bufry layer is removed with a transfer pipette and washed threetimes in “HHF”, composed of Hank's balanced salt solution (HBSS; LifeTechnologies, Gaithersburg, Md.), containing 5% fetal calf serum (FCS,CSL Limited, Victoria, Australia).

TNAP+ were subsequently isolated by magnetic activated cell sorting aspreviously described (Gronthos et al., 2003; Gronthos et al., 1995).Briefly, approximately 1-3×10⁸ BMMNC are incubated in blocking buffer,consisting of 10% (v/v) normal rabbit serum in HHF for 20 minutes onice. The cells axe incubated with 200 μl of a 10 μg/ml solution of STR-3mAb in blocking buffer for 1 hour on ice. The cells are subsequentlywashed twice in HHF by centrifugation at 400×g. A 1/50 dilution of goatanti-mouse γ-biolin (Southern Biotechnology Associates, Birmingham, UK.)in HHF buffer is added and the cells incubated for 1 hour on ice. Cellsare washed twice in MACS buffer (Ca²⁺- and Mn²⁺-free PBS supplementedwith 1% BSA, 5 mM EDTA and 0.01% sodium azide) as above and resuspendedin a final volume of 0.9 ml MACS buffer.

One hundred μl streptavidin microbeads (Miltenyi Biotec; BergischGladbach, Germany) are added to the cell suspension and incubated on icefor 15 minutes. The cell suspension is washed twice and resuspended in0-5 ml of MACS buffer and subsequently loaded onto a mini MACS column(MS Columns, Miltenyi Biotec), and washed three times with 0.5 ml MACSbuffer to retrieve the cells which did not bind the STRO-3 mAb. Afteraddition of a further 1 ml MACS buffer, the column is removed from themagnet and the TNAP-positive cells are isolated by positive pressure. Analiquot of cells from each fraction can be stained with streptavidm-FITCand the purity assessed by flow cytometry. STRO-3 mAb was found toidentify a minor subpopulation of-BMMNCs (<1%).

Primary cultures are established from the MACS isolated TNAP+ cells byplating in α-MEM supplemented with 20% fetal calf serum, 2 mML-glutamine and lOQμm L-ascorbate-2-phosphate as previously described(Gronthos et al., 1995).

Example 5 Human Bone Marrow Cells Selected by STRO-3 mAb Give Rise toCFU-F Materials and Methods Magnetic-Activated Cell Sorting (MAGS)

To evaluate the CFU-F outgrowth potential OfTNAP++ cells, MACS sortingwas used to separate TNAP+ and TNAP− cells from the bone marrow. Thiswas performed generally as previously described (Gronthos and Simmons,1995; Gronthos et al., 2003) but using the STRO-3 mAb. In brief,approximately 1-3×10⁸ normal human bone marrow mononuclear cells wereincubated with STRO-3 supernatant, anti-IgG-biotii-, streptavidinmicrobeads and finally streptavidin. FITC (Caltag Laboratories,Burlingame, Calif.) before being separated on a Mini MACS magneticcolumn (Miltenyi Biotec Inc., Auburn, Calif.) according to themanufacturers instructions.

Fluorescence-Activated CellSorting (FACS)

CFU-F outgrowth capacity was also examined on STRO-3 selected cellssorted on the basis of +ve or −ve expression of STRO-1.

This was performed as previously described (Gronthos and Simmons, 1995;Gronthos et al., 2003). In brief, approximately 1-3×10⁸ normal humanbone marrow mononuclear cells were sequentially incubated with STRO-1supernatant, anti-IgM-biotin, microbeads and finally streptavidin FITC(Caltag Laboratories, Burlingame, Calif.) before being separated on aMini MACS magnetic column, (Miltenyi Biotec Inc, Auburn, Calif.)according to the manufacturers instructions.

The STRO-1⁺ MACS isolated cells were labelled with streptavidinconjugated FITC then incubated either purified STRO-3 mAb or isotypecontrol 1B5 (10 μg/ml) for 30 minutes on ice, washed and incubated withphycoerythrin (PE) conjugated goat anti-mouse IgG antibody ( 1/50;Southern Biotechnology Associates, Birmingham, Ala.) for an additional20 minutes on ice. Cells were sorted using a FACStar^(PLUS) flowcytometer (Becton Dickinson, Sunnyvale, Calif.). TheSTRO-1^(bri)/STRO-3⁺ or STRO-1^(bri)/STRO-3″ cells were cultured inalpha-Modification of Eagle's Medium supplemented with 20% fetal calfserum, L-glutamine 2 mM, ascorbate-2-phosphate (lOOμM) to initiateprimary culture in 5% CO₂, at 37° C. humidified atmosphere.

Results and Discussion

Experiments were designed to examine the potential of using STRO-3 mAbas a single reagent for isolating cells for CFU-F outgrowth (FIG. 8),MACS isolation based on TNAP expression revealed that clonogenic CFU-Fwere only detected in the TNAP⁺ BMMNC fraction.

Given that STRO-3 (IgGi) is a different isotype to that of STRO-I (IgM),the ability of STRO-3 to identify clonogenic CFU-F was assessed bytwo-colour FACS analysis based on its co-expression-with STRO-1⁺ cellsisolated using the MACS procedure (FIG. 9). STRO-3 mAb demonstrated aunique binding pattern, reacting with a subset of the STRO-1⁺ BMMNCfraction which expressed the STRO-1 antigen at high levels(STRO-1^(bright) fraction), effectively isolating and enriching for theMPC population (Table 2). Furthermore, the mAb STRO-3 failed to reactwith CD34 positive haemopoietic stem cells in human adult bone marrowaspirates (data not shown).

TABLE 2 Enrichment of human bone marrow cells by dual-colour FACSanalysis based on the co-expression of the cell surface markers STRO-Iand TNAP (refer to FIG. 9). FACS sorted cells were cultured understandard clonogenip conditions in alpha MEM supplemented with 20% PCS.The data represents the mean number of day 14 colony-forming cells(CFU-F) per 10⁵ cells plated ± SE (n = 3 different bone marrowaspirates). These data suggest that human MPC are exclusively restrictedto the TNAP positive fraction of BM which co-express the STRO-I antigenbrightly. Frequency of Enrichment Bone Marrow Fraction CFU-F/10⁵ Cells(Fold Increase) Unfractionated BMMNC  11.0 ± 2.2 1.0TNAP+/STRO-1^(bright) 4.511 ± 185 410 TNAP+/STRO-1^(dull/int) 0.0 0.0

Example 6 Phenotype of STHO-3 mAb Selected Cells Before and afterCulture Expansion Materials and Methods

Single color flow cytometry was performed essentially as described inGronthos et al. (1999), Briefly, cultures of cells at each passage wereliberated by trypsin/EDTA and subsequently incubated for 30 min on icein blocking buffer. Approximately 1×10⁵ cells were washed as describedabove, and resuspended in 200 μl of primary antibody or antibodies for 1hr on ice. The primary antibody consisting of saturating concentrationsof the mouse IgM monoclonal antibody STRO-1 and/or a mouse IgGmonoclonal antibody to human CC9 and STRO-3 were used. Other antibodiesused included mAbs that bind CD45, CD34 and 3G5.

The mouse isotype IgM and IgG negative control mAbs were treated underidentical conditions. After the cells were gashed, second label(s) wereadded in a final volume of 100 μl consisting of goat anti-mouse IgMμ-chain specific-FITC ( 1/50 dilution) and either goat anti-mouse IgGγ-specific-PE ( 1/50 dilution) or anti-rabbit Ig-specitlc-PE ( 1/50dilution.) (Southern Biotechnology Associates). The cells were incubatedfor 45 min on ice, washed twice and fixed in FACs FIX (PBS supplementedwith 1% (v/v), 2% (w/v) D-glucose, 0.01% sodium azide). The cells werethen analysed on an Epics®-XL-MCL flow cytometer (Beckman Coulter,Hialeah, Fla.). The dot plot histogram represents 5×10⁴ events collectedas listmode data.

Results and Discussion

As can be seen in FIG. 10, immunoselection of human bone marrowmononuclear cells using a magnetically labelled STRO-3 mAb results hiisolation of a population of cells characterised by high levels of TNAPand CD45 surface antigen expression (FIG. 10), with approximately 90% ofthe TNAP+ cells co-expressing CD45. In contrast, less than 1% of theTNAP+ enriched cells isolated using the STRO3 mAb expressed thehematopoietic stem cell marker CD34. In addition, no more than 5% of theTNAP+ cells enriched using methods of the invention co-expressed any ofthe markers previously used to isolate MPC, including STRO-1, CC9/CD146,and 3G5.

Following culture-expansion, the TNAP+ cells enriched using methods ofthe invention demonstrated a stable phenotype.-that differed from theenriched and freshly-isolated cells, FIG. 11. Culture expanded STRO-3selected cells at both early (passage 2) and late (passage 5) passageswere found to express homogeneously high levels of CC9/CD146 and HLAclass I molecules, but were uniformly negative for CD45, HLA class II,CD14, CD 19, CD3, CDlla-c, CD31, CD86 and CDSO. Strikingly, while TNAPsurface expression as detected by STRO-3 mAb was uniformly positive inearly culture passages (passage 2), this was negative at later culturepassages (passage 5).

In contrast to progressive loss of STRO-3 reactivity following cultureexpansion, these TNAP+ cells enriched by STRO-3 selection demonstratedprogressive increase in surface expression of the STRO-1 antigen, FIGS.11 and 13. By passage 2 approximately 84% of the cells were STRO-Ipositive in comparison to the IgM isotype control, and approximatelyhalf (52%) expressed STRO-1 brightly (as defined by a 2 log magnitudehigher expression of STRO-1 surface expression than STRO-1 negativecells). By passage 5, while most of the cells remained STRO-1 positive(approx. 69%), a lower proportion of cells expressed STRO-1 brightly(approx. 21%). This indicates that initial culture of STRO-3 selectedTNAP+ cells results in upregulation of the STRO-I antigen, presumablyreflecting proliferation without spontaneous differentiation, whileongoing culture results in dowπregulation of STRO-1 antigen density frombright to intermediate expression.

In marked contrast, immunoselectioα of human bone marrow mononuclearcells using a magnetically labelled STRO-1 mAb results in isolation of apopulation of cells characterised by high (approximately 50%) STRO-1expression, and absence of CD45 expression (FIG. 12, andWO/2004/085630). Despite this high-level of initial STRO-1 expression,culture expansion of STRO-1 selected cells results in a progressivedecrease in STRO-1 expression by passages 4 to 6. These reduced STRO-1levels following culture-expansion, are significantly reduced relativeto both the starting population, and the culture-expanded TNAP+ enrichedcells by STRO-3 selection at the same passages 4 to 6 (Figure H).

Together, these results show that the TNAP+ enriched population bySTRO-3 selection is distinct from tho STRO-1+ enriched population interms of phenotypic characteristics both when initially freshly isolatedand following culture-expansion.

Example 7 Differentiation of TNAP+ Cells—Adipogenesis Materials andMethods Adipogenic Assay Procedure

Preparation of Adipogenic Induction Medium: Adipogeric Induction Mediumshould be used once the cells have become 100% confluent (approximately5-13 days). Prepare the medium before the cells become confluent.

-   L Decontaminate the external surfaces of the Adipogenic Induction    Medium (PT-3 102B) and the following SingleQuots® with 70% v/v    ethanol or isopropaπol:    -   a. h-Insulin (recombinant)    -   b. L-Glutamiηe    -   c. MCGS    -   d. Dwcamethasone    -   e. Indomethacin    -   f. IBMX (34sobuty-1-methyl-x.tn.thme)    -   g. Pen/Steep-   2: Aseptically open the above SingleQuots and add the contents to    the 175 ml of Adipogenic Induction Medium.-   3. Rinse each. SingleQuot vial with the medium,-   4. Use supplemented medium for the adipogenie induction of cells    only. Store at 2° C. to 8° C. in the dark until needed.

Prepare Adipogenie Maintenance Medium as follows:

-   1. Decontaminate the external surfaces of the Adipogenie Maintenance    Medium (PT-3102A) and the following SingleQuots with 70% v/v ethanol    or isopropaool:    -   a. h-Insulin (recombinant)    -   b. L-Glutamine    -   c. MCGS    -   d. Pen/Strep-   2. Aseptically open the above SingleQuots and add the contents to    the 175 ml of Adipogenie Maintenance Medium.-   3. Rinse each SingleQuot vial with the medium.-   4. Store supplemented Adipogenie Maintenance Medium at 2° C. to    8° C. in the dark until needed.

Adipogenesis Culture Protocol:

-   1. Plate 2.1×10⁴ STRO-3 mAb selected cells per cm² of tissue culture    surface area in 0.2 to 0.3 ml of MSCGM per cm² of tissue culture    surface area. For example: 2×10⁵ cells in 2 rol medium per 9.6 cm²    well of a 6 well plate. Inoubate the cells at 37° C., in a    humidified atmosphere of 5% CO2.-   2. Feed the cells every 2-3 days by completely replacing the medium    with fresh MSCGM until the cultures reach confluence (5-13 days).    The cells must be confluent, or post confluent, for optimal    Adipogenie differentiation.-   3. At 100% confluence, three cycles of induction/maintenance will    stimulate optimal Adipogenie differentiation. Each cycle consists of    feeding the cells with supplemented Adipogenesis Induction Medium    and culture for 3 days (37° C., 5% CO₂) followed by 1-3 days of    culture in supplemented Adipogenie Maintenance Medium. Feed    non-induced control cells with only supplemented Adipogenie    Maintenance Medium on the same schedule. Adipogenie cells are    delicate and care should be used to avoid disrupting the numerous    lipid vacuoles in the cells. Do not let the cells dry out when    changing medium.-   4. After 3 complete cycles of induction/maintenance, culture the    cells for 7 more days in supplemented Adipogenic Maintenance Medium,    replacing the medium every 2-3 days.-   5. The extent of Adipogenic differentiation may be noted by    microscopic observation of lipid vacuoles in the induced cells. To    document the Adipogenic differentiation, cultures may be stained    with AdipoRed. Non-induced cells will have few, if any, lipid    vacuoles.-   6. Cultures of unfixed cells may be used for assays requiring    adipocytes.

AdipoRed™ Assay for In Vitro Adipogenesis

Protocol for 6-, 12-, 24- and 48-well plates:

-   -   1. Seed cells at 30,000/cm² and culture and differentiate the        cells as described above, using appropriate volumes of cell        culture media.    -   2. Immediately prior to the assay, rinse each plate with PBS,        and add AdipoRed, using the volumes in Table 3.

TABLE 3 Volumes for AdipoRed ™ assay. Rinse Final volume Volume ofvolume/well of PBS/well AdipoRed/well 6-well plate 2 ml 5 ml 140 μl12-well plate 1 ml 2 ml 60 μl 24-well plate 1 ml 1 ml 30 μl 48-wellplate 0.4 ml 0.4 ml 12 μl 96-well plate 0.2 ml 0.2 ml 5 μl

-   -   3. After addition of the AdipoRed, the best mixing of the        reagent is obtained by pipetting 50% of the contents of each        well up and down two times (three times for 6-well plates). It        is important to obtain a homogeneous dispersion of the blue        AdipoRed reagent Be very careful not to touch the tip of the        pipette to the cell monolayer or remove cells from the well        surface.    -   4. After 10 minutes, place the plate in the fluorimeter and        measure the fluorescence with excitation at 485 run and emission        at 572 nm. If the fluorimeter does not have the appropriate        filters, the settings used for the common fluorophore        fluorescein (excitation 485 ran; emission. 535) can be used.

Results and Discussion

Two lots of cells were assayed for differentiative capacity. The amps ofcells were labeled:

2242A 2070C P.2o P.2o −30E6 cells −3QE6 ceils 102NOV2005 08NOV2005

The results are provided in FIG. 14 and show that cells selected withSTRO 3 mAb are capable of differentiating into adipocytes.

Example S Differentiation of TNAP+ Cells—Osteogenesis Materials andMethods Osteogenic Assay Procedure

Prepare Osteogenic Induction Medium as follows:

-   1. Decontaminate the external surfaces of the Differentiation Basal    Medium—Osteogenic and the following SingleQuots with 70% v/v ethanol    or isopropanol:    -   a. Dexamethasone    -   b. L-Glutamine    -   c. Ascorbate    -   d. Pen/Strep    -   e. MCGS    -   f. β-Glycerophosphate-   2. Aseptically open the above SingteQuots and add the contents to    the 185 ml of Differentiation Basal Medium—Osteogenic.-   3. Rinse each SingleQuot vial with the medium.-   4. Store the supplemented Osteogenic Differentiation Medium at 2° C.    to 8° C. in the dark until needed.

Osteogenesis Culture Protocol:

-   1. Plate 3.1×10³ STRO-3 mAb selected cells per cm² of tissue culture    surface area in 0.2-0.3 ml of MSCGM per cm² tissue culture area. For    example: 3×10⁴ cells in 2 ml medium per 9.6 cm² well of a 6 well    plate.-   2. Allow the cells to adhere to foe culture surface for 4 to 24    hours in MSCGM at 37° C., in a humidified atmosphere of 5% CO₂.-   3. Induce Osteogenesis by replacing the MSCGM with Osteogenesis    Induction Medium.-   4. Feed the induced cells every 3-4 days for 2-3 weeks by completely    replacing the medium with fresh Osteogenesis Induction Medium. Feed    non-induced control cells with MSCGM on the same schedule.-   5. Osteogenic induced cells will show changes in cell morphology,    from spindle shaped to cuboidal shaped, as they differentiate and    mineralize. Gaps may form in the post confluent cell layer and cells    may begin to delaminate from culture surface. If this de-lamination    is observed, proceed immediately to analysis of osteogenic    differentiation as indicated by calcium deposition, or use the    induced cells for other assays requiring osteocytes.-   6. For calcium deposition assays, harvest cells by rinsing them in    calcium free PBS, then scraping cells from the culture surface in    the presence of 0.5M HCl. Assay the extracts from osteogenic induced    cultures for calcium content and compare to extracts from    non-induced control cells.

Calcium Deposition Assay for In Vitro Osteogenesis Materials:

DPBS, without calcium or magnesium—Cambrex catalog #17-5 16Q

-   -   0.5NHC1    -   Calcium (CPC) Liquicolor kit—Stacbio Laboratory catalog        #0150-250    -   Induced Osteogenic cultures    -   Plate reader or spectrophotometer

Procedure:

-   -   Aspirate all culture medium from each well of a 6-well culture        plate that contains induced or control cells to be tested.    -   Rinse the cells in the plate by adding 1 ml of PBS to the side        of each well, being careful to not dislodge the cells.    -   Aspirate off the PBS and re-rinse, as above.    -   Aspirate the second wash and add 0.5 ml of Q.5N HCl to each        well.    -   Scrape the cells off of the surface using a cell lifter and        transfer the cells and HCl to a polypropylene tube (1.5 ml        Eppendorf tube or any 2-5 ml polypropylene tube with a tight        fitting cap).    -   Add an additional 0.5 ml of 0.5N HCl to each well to recover any        cells remaining in the well, and transfer this to the        appropriate tube.    -   Samples may be capped tightly and stored at −20° C. for one        month if they are not to be tested immediately.    -   Extract the calcium from the cells by snaking the tubes on an        orbital shaker for 3-24 hours at 4° C. If using frozen samples,        allow extra time for samples to thaw.    -   Centrifuge the sample tubes at 500 g for 2 minutes.    -   Carefully collect the supernatant with extracted calcium,        without disrupting the pellet, and transfer to a new tube,    -   Following the instructions provided in the Stanbio Laboratory        Calcium (CPC) Liquicolor kit, prepare a standard curve with the        calcium standard and deteππine the amount of calcium in each        control and osteo-induced sample.    -   Sample and assay reagent volumes may be adjusted to fit        microliter plates (200 μl) or spectrophotometer cuvettes (2 ml).    -   10 μl-100 μl of sample is used for each calcium determination.        Unused sample extract may be re-frozen for future re-assay.

Results and Discussion

The results are provided in FIG. 15 and show that cells selected withSTRO-3 mAb are capable of differentiating into osteocytes.

Example 9 Differentiation of TNAP++ Cells—Chondrogenesis, Adipogenesisand Osteogenesis

Choπdrogenic differentiation was assessed in aggregate cultures treatedwith 10 ng/ml TGF-β3 as previously described (Gronthos et al., 2003).Alcian Blue demonstrated that STRO-3 mAb selected cells are capable ofproducing proteoglycan (FIG. 16C), and thus chondroctyes.

STRO-3 mAb selected cells were also differentiated into functionalosteoblasts, following 3 weeks culture in αMEM supplemented with 10%FCS, 100 μL-ascorbate-2-phosphate, dexamethasone 10-7 M and 3 mMinorganic phosphate. Mineral deposits were identified by positiveAlizarin Red staining (FIG. 16A).

Similarly, adipogenesis was induced in the presence of 0.5 mM methylisobutylmethylxanine, 0.5 μM hydrocortisone, and 60 μM indomethacinas previously described (FIG. 16B). Oil Red O staining demonstrated thepresence of lipid-laden fat cells.

Example 10 Transplantation of Expanded Human STRO-3 mAb Selected CellsInduce New Bone Formation In Vivo

Approximately 5.0×10⁶ ex vivo expanded cells derived from STRO-3 mAbselected bone marrow cells were mixed with 40 mg ofhydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer Inc,Warsaw, Ind.) and then transplanted subcutaneously into the dorsalsurface of 6-week-old immunocompromised NOD/SCID mice (ARC, Perth, WA₅Australia) fox eight weeks as previously described ((Gronthos et al.,2003). These procedures were performed in accordance to specificationsof art approved animal protocol (University of Adelaide Ethics Number M19/2005), Harvested implants were fixed in 4% paraformaldehyde, thendecalcification with 10% EDTA solution, before being embedding intoparaffin. A representative cross section of a 8 week old transplantstained with H&E is shown. Histological examination demonstrated thepresence of new bone formation (FIG. 16D).

Example 11 STRO-3 raAb Selected Cells are useM in Bone Repair Materialsand Methods Tibia Critical Sized Defect

With the sheep in dorsal recumbency, the wool and hair was removed fromabove the left bind limb (mid femur) down to the foot The skin over thetibia was prepared for aseptic surgery using alternating scrubs ofpovidone-iodine (Betadine) and alcohol. The limb was draped for asepticsurgery. Peri operative antibiotic prophylaxis (Ancef; 1 gmpreoperatively, 1 gm mid-surgery and 1 gm following wound closure,intravenously) began at this point. A 6-cm skin incision extendingthrough the periosteum was made over the medial diaphysis of the tibia.The periosteum and overlying soft tissues was bluntly elevatedcircumferentially. A 5-cm segmental defect was created mid-diaphysiswith two osteotomies using an oscillating saw under constant coolingwith saline solution.

The defect was repaired using a locking inteπnedullary nail. Forinsertion of the nail, a longitudinal incision, just medial to themidline, was made over the left knee (stifle) joint with the knee in aflexed position. The joint capsule was split, the patellar tendonretracted laterally, -aid the centre of the tibial plateau dissectedfree of adipose tissue within the joint. A 6-mm entry portal was createdby a drill- and the diaphysis of the tibia reamed with hand reamersuntil the nail can be inserted press-fit If necessary, the distal tibialmetaphysis was reamed with an 8-mm drill so the distal part of the nailcould be inserted manually. The nail was inserted with the use of theinsertion handle and the driving head connected to the proximal end ofthe nail. Proximal and distal interlocking was performed with proximaland distal aiming devices.

Administration 1 Culture Expanded STRO-3 mAb Selected Cells and HA/TCPCarrier to Sheep Tibia Critical Sized Defeat

The defect was packed with an HA/TCP carrier or HA/TCP+ STRO-3 mAbselected culture expanded cells and tested at varying concentrationsdepending on the treatment group (25M, 75M or 225M) following briefincubation in the OR setting. The soft tissues was then closed over thedefect to ensure containment of the carrier and cells.

Animals had plain radiographs taken lateral and cranio-caudal (AP) undergeneral anaesthesia. Radiographs were taken at the followingtintepoints: Day.O (surgery) and 1, 2, and 3 months.

Radiographs were interpreted according to the following criteria:

% callus bridge was the summed measure of the amount of mineralizedtissue extended into the defect area both proximal and distal anddivided it by the total defect length.

Fusion was characterized by a scoring system below:

0 (no fusion);1 (moderate fusion);2 (robust interconnected fusion mass).

Spine Fusion Procedure

Sheep were anesthetized and wool was removed from the dorsal lumbarregion of the sheep and positioned in sternal recumbency on theoperating table. The lumbar region was prepared for aseptic surgery withmultiple scrubs of povidone-iodine alternated with isopropyl alcohol.The area was draped and local anesthesia (Lidocarae), was infiltratedalong dorsal approach to L4 and L5 dorsal to the spinous processes.

Approach to the transverse processes: A 20 cm skin incision was made andthe paraspinal muscles will be dissected off the spinous processes andlaminae. Facet joints and transverse processes between. L3 and L4 willbe exposed.

Instrumentation and Spine Fusion Technique: The transverse processes ofL3 and L4 was decorticated bilaterally. The HA/CP graft substitute(carrier) or carrier+ allogeneic cells or autograft was placed betweenthe transverse processes. At this point in the surgery, the sheepundergo transpedicular screw fixation using screws and rods(Medtromc-Sofamor-Danek; CD-Horozon, M8 fixed screw head system). Thesurgical site was closed routinely.

After 4 months, all sheep were humanely euthanized and immediately afterremoval of the connecting rods, the explanted spines subjected to CTscans and plain radiographs and faxitron analysis.

Mechanical Testing Spine

Immediately following euthanasia, intact lumbar spines were harvestedand immediately prepared for mechanical testing, A four vertebraeconstruct consisting of the two affected (fused) vertebrae as well as anadditional vertebral level above and below the level of the fusion wasisolated from the lumbar spine. These isolated specimens were denuded ofall peri-spinal soft tissues, with care taken to preserve anyligamentous and facet capsule architecture. Several screws were drilledinto superior endplate of most cephalad vertebra and the inferiorendplate of the caudad vertebrae were coupled to metal potting fixtures,with the screw-vertebra construct secured using polymethylmethacrylate(PMMA). Specimens were kept moist during the entire preparation andtesting procedure.

Kinetic Analysis—Load Application and Range of Motion Determination

The specimen was attached to a custom-designed spinal testing fixture.The testing fixture wag coupled to a standard servohydraulic testingframe (MTS) and, using a system of pulleys and tensioned wires, appliespure moments to the specimen in flexion/extension, right and leftlateral bending, and right and left axial rotation. Loads were appliedup to a maximum of 5 N-m. Specimens were pre-conditioned for threecycles and data will be collected on the fourth cycle.

Load-dependent three-dimensional displacements were calculated using theprinciples of stereophotogrammetry. Three non-collinear markers will beattached to both the inferior and superior potting fixtures and the twolevels that are involved with the fusion. Three high-resolution cameras(Vicon Peak, Centennial, Colo., USA) were used to detect the lightreflected by these markers, and the collected data was processed withcustom-designed software (Spinal Flexibility Testing Software, MFLEX) todetermine the appropriate intervertebral angles across the fusion mass.The resulting data provided both neutral zone and range of motion dataacross the involved levels and the adjacent segments for all threebending planes.

Statistical Analysis

Statistical significance in the aforementioned parameters betweentreatment groups is performed using a standard one-way ANOVA withFisher's least-significant-difference PLSD post hoc test for multiplecomparisons (StatView, SAS Institute Inc. Cary, N.C., USA), p-valuesless than 0.05 will be considered statistically significant.

Animals had plain radiographs taken lateral and cranio-caudal (AP) undergeneral anaesthesia, Radiographs were taken at the following timepoints:Day 0 (surgery) and 1, 2, 3 and at 4 months. Additionally, faxitronanalysis was performed at time of sacrifice using mammography film.

Radiographs were interpreted according to the following criteria:

0 (no fusion);1 (moderate fusion);2 (robust interconnected fusion mass).

Results and Discussion

FIG. 17 shows STRO-3 mAb selected culture expanded allogeneic adultmultipotential cells result in significant spinal fusion followingadministration with an HA/TCP carrier in an ovine transpedicular screwfixation spine model compared to control alone or autograft asdetermined by x-ray analysis. Significant spinal fusion was observed asearly as 3 months and continued to increase at 4 months. This effect didnot appear to be dose dependent as even the lowest dose resulted insignificant spinal fusion compared to control carrier and autograft.

FIG. 18 shows STRO-3 mAb selected culture expanded allogeneic adultmultipotential cells administered with an HA/TCP carrier in ovinetranspedicular screw fixation spine model demonstrate robust spinalfusion compared to carrier controls at time of sacrifice when theinstrumentation has been removed and the area assessed by fexiironanalysis using mammography film. All doses of multipotential cellsdemonstrate density of spinal fusion comparable to autograft standard ofcare.

FIG. 19 shows STRO-3 mAb selected culture expanded allogeneic adultmultipotential cells administered with an. HA/TCP carrier in ovinetranspedicular screw fixation spine model demonstrate fusion that ismechanically comparable to autograft controls. All doses ofmultipoteπial cells resulted in flexion, lateral extension and torsionalrange of motion mechanical loads characteristic of fused bone.

FIG. 20 shows STRO-3 mAb selected culture expanded allogeneic adultmultipotential cells administered with an HA/TCP carrier in ovinecritical sized 5 cm defect tibia model resulted in early bone growth ina dose dependent manner. 225M cells combined with HA/TCP carrierresulted in over 60% callus bone formation as early as 3 monthsfollowing injury.

FIG. 21 shows STRO-3 mAb selected culture expanded allogeneic adultmultipotential cells administered with an HA/TCP carrier in ovinecritical sized 5 cm defect tibia model resulted in increased union ratescompared to carrier alone control. Only the 75M and 225M cell doseresulted in union as defined by x-ray analysis at 3 months.

These results show that expanded cells of the invention are able toenhance bone repair.

Example 12 STRO-3 mAb Selected Adult Multipotential Cells ImproveCardiac Function Materials and Methods Thoracotomy Procedure

For sheep, chest and upper abdomen was clipped, prepped with soap andwater and painted with betadine solution which is allowed to dry. Thesurgical fields were draped with sterile drapes and all persons at theoperating table were fully gowned, masked, gloved and capped. Allthoracic operations are done through a left thoracotomy. The smallestpossible incision was used and the 3^(rd), 4^(th) or 5^(th) interspaceis entered. Skin incisions were made with scapel. Subcutaneous tissueand muscles are usually divided with cautery to improve hemostasis. Thepericardium was opened and the heart supported in a pericardial cradle.An epicardial echocardiogram was performed under sterile technique.Polypropylene (#0) sutures are used to ligate the appropriate coronaryarteries. The anteroapical infarction model has been well establishedpreviously in this animal model. Briefly, suture ligation of the distal⅓ of the left anterior descending artery (LAD) along with ligation ofthe second diagonal coronary artery branch (D2) will uniformly create ananteroapical Infarct comprising approximately 20-25% of the leftventricle; this technique consistently and reliably produces injurywhich leads over time to ventricular remodeling and congestive heartfailure (CHF). Thoracotomy wounds were closed with running 3-0 Vicrylsuture, Skin was closed with a subcuticular suture of 3-0 Vicryl. Priorto chest closure an intercostal nerve block at the surgical site wasperformed with bupivicaine (5 cc of 0.25% solution). A chest tube wasplaced in the left pleural space and placed on 20 cr αwater suctiondrainage until the animal is extubated.

Animals were fully monitored while under anesthesia during the procedure(eg, BP—arterial line, Cardiac output—Swan-Ganz/conductance catheter).Animals are carefully watched in our laboratory for several hours postextubalion until fully awake and standing—They are closely watched forthe next 6-24 hours for any arrhythmias or signs of low cardiac outputand monitored and treated for pain control, fluid retention or lack ofappetite as needed.

Additional thoracotomy were performed in nude rats. Leftanteriodecending artery ligation was performed under general anesthesiaand animals were subsequently injected with cells in the perinfarctregion, pericardium and wounds sutured and animals were allowed tosurvive for 2 weeks at which time they were sacrificed.

Administration of Adult Multipotential Cells

For sheep in intro expanded STRO-3 TOAb selected bone marrow cells orcontrol Profreeze media was thawed in a 37 degree waterbatch. The 4 mlvial was then swabbed with alcohol and an angiocath needle syringe wasthen used to aspirate ImI volumes and transported into 4 1 ml syringes.A total of approximately 3.5 mls of the 4 mls was recovered. Each 1 mlsyringe was then fitted with a 27 gauge needle and 0.2 ml was injectedaround the perinfarct borderzone via approximately 16-20 injections.

For rats, 0.2 ml of media containing one million cells was injected by27 gauge needle at the perinfarct border zone.

Echocardiography Laparotomyfor Transdiaphragmatic QuantitativeEchocardiography

For sheep, at approximately baseline, immediately post-infarction, weekfour post-infarction each animal underwent laparotomy under full generalanesthesia (isoflurane) to perform transdiaphragmatic quantitativeechocardiography. A laparotomy is required because it is not possible toget adequate transthoracic or transesophageal echocardiographio imagesfor quantatative analysis in sheep. These animals have enormous lungsthat wrap around the heart entirely. The air in the lungs degrades theimages substantially. During these studies hemodynamics such as bloodpressure, heart rate and cardiac output were carefully monitored.

Specifically, the upper abdomen was clipped, prepped with soap and waterand painted with betadiπe solution which is allowed to dry. The surgicalfield was draped with sterile drapes and all persons at the operatingtable were fully gowned, masked, gloved and capped. Because ofoverlapping lungs echocardiograms were taken from a subdiaphragmaticview, The initial incision was made with a scalpel in the midline andcarried through the subcutaneous and muscular layers with a cautery. Theperitoneal cavity was opened. The echo probe was introduced under thediaphragm within a sterile plastic bag and all studies are done understerile conditions. The incision is closed with simple interrupted0-ρrolene suture through both the peritoneum and posterior fascia. Thesubcutaneous tissue was closed with 3-0 running Vicryl. The skin isclosed with 3-0 subcuticular Vicryl.

For rats, 2D echocardiography was used to measure to systolic anddiastolic volume parameters.

Data Analysis

All images were analyzed off-line. All measurements were made at endsystole, identified as the frame at which LV cavity area was smallest.All plots representing three-dimensional renderings were created usingTecplot (Version 10; Amtec Engineering, Bellevue, Wash.). Spline surfacefits and Gaussian curvatures were calculated in Matlab. AU measurementsare presented as means SD. Comparisons are made between baseline andpostinfraction using paired t tests. The image processing and dataanalysis performed for the 2DCE data in both long and short axis hasbeen described previously. Briefly, the endocardialcurvature (K) and theventricular wall thickness (h) were measured in the borderzone beforeand 1 hour after infarction. All 3DCE rotational cross-sectional imageswere analyzed as follows. Endocardial and epicardial contours weretraced (UTHSCSA ImageTool; Department of Dental Diagnostic Science,University of Texas Health Science Center, San Antonio, Tex.) by anechocardiography technician unaware of the hypotheses of the study.

The endocardium and epicardium were reconstructed at end systole bytracing each in every individual rotational cross-section. At eachendocardial and epicardial location, the presence or absence ofmyocardial perfusion was determined; thereby, perfusion status wasregistered with LV geometry, allowing precise and unambiguousdetermination of borderzone myocardium. The cross-sectional data werethen combined to recreate a threediroensional representation of the LVendocardial and epicardial surfaces, indexed by perfusion status. Theendocardial and epicardial surfaces were fit using a smoothingthin-plate spline (x), and the characteristics of this surface,including spatially resolved Gaussian curvature, were calculated.

Results and Discussion

FIG. 22 shows the effects of human STRO-3 mAb selected cells following 5passages directly injected into hearts of 5 rats 24 hours after acuteligation of the left anterior descending coronary artery. At two weeksthe cells induce approximately 50% greater fractional area changecompared with injection of control medium alone.

FIG. 23 shows the effects of allogeneic sheep STRO-3 mAb selected cellsfollowing 5 passages directly injected into sheep hearts immediatelyafter acute ligation of both diagonal coronary arteries. At four andeight weeks the animals treated with STRO-3 selected cells demonstratesignificantly greater ejection fraction (A), and significantly lowerdiastolic (B) and systolic (C) volumes, compared with animals treatedwith control medium alone.

These results show that expanded cells of the invention are able toimprove cardiac function.

Example 13 Use Of Human STRO3-Selected and Culture-Expanded TNAFEnriched Cells in Human Patients in Need Qf (1) Bone Regeneration, or(2) Cardiac Functional Recovery/Increase in Blood Vessel FormationMaterials and Methods

Standard operating protocols of Cell Therapies Pty Ltd (affiliated withPeter MacCullum Institute of Cancer Research Melbourne, Australia) wereused on culture expanded STRO-3 mAb immunoselected TNAP+ cells fromhuman BM, enabling their subsequent in vivo use in patients in need ofeither bone regeneration or cardiac function/blood vessel formation.

Bone Marrow Aspiration Procedure

1. Bone Marrow (BM) is routinely taken from two or more sitesapproximately ½ to 1 cm apart on the back of the iliac crest (hip bone).

2. An injection of local anaesthetic is given in the skin over the hipto anaesthetise the skin area. A small cut is made in the skin and aneedle is placed into the bone.

3. 5-20 ml of marrow is aspirated the needle is withdrawn and reinsertedthrough the same skin incision into a different part of the bone, awayfrom the previously aspirated area until 40 ml of marrow has beencollected.

4. BM is routinely aspirated into Lithium-Heparin containing tubes,although other anti-coagulants are acceptable. It is preferable that themarrow aspirate is processed within 1 hour of collection, as describedbelow.

Bone Marrow Mononuclear Preparation—Density Gradient Separation

All techniques are performed in a Biological Safety Cabinet Class 11

1. A 40 ml aspirate of BM will usually be received in 4 tubes (approx.10 ml/tube).

2. Pool all the fractions of BM into a 50 ml tube (Falcon, BectonDickinson) to ensure equal mixing. Divide BM volume into equal amountsinto two 50 ml tubes. Add an equal volume of blocking buffer.

3. Perform a white cell estimation using white cell fluid (WCF). Assesscell number (pre-processing count).

4. Using a 70 mm cell strainer (Falcon, Becton Dickinson), strain thediluted BM into two 50 ml centrifuge tubes to remove any small clots andbone fragments.

5. Place 3 mls of Ficoll-Hypaque (Lymphoprep) solution in the bottom oftea “round bottom” 14 ml polystyrene tubes (Falcon, Becton Dickinson).

6. Carefully overlay lymphoprep with 7.5 mls of BM.

7. Centrifuge tubes at 400×g (1400 rpm) for 30 mins at RT. Ensure thatthe centrifuge brake is off

8. With a sterile cannula, vacuum aspirate media until approximately _cm above the leucocyte band (buffy coat). Carefully collect themononuclear layer with a disposable plastic Pasteur pipette and poolinto a 50 ml tube.

9. Dilute cells to 40 ml with wash buffer and centrifuge sample at 400×g(1400 rpm) for 10 amiss with the break on high.

10. Aspirate the buffer until just above the cell pellet Vortex the tubeand add 50 ml HHF. Repeat step 9.

Magnetic Activated Cell Sorting (MACS) of TNAP Positive CeOs

1. Prior to immunolabeling, BMMNC (approximately 1-2×108 cells) areresuspended in 0.5 ml blocking buffer and incubated for 30 minutes onice to block possible Fc receptor-mediated binding of antibodies.

2. Five hundred micro liters of STRO-3 mAb previously diluted to aconcentration of 10 mg/ml in blocking buffer, is added to the BMMNC andincubated for 60 minutes at 4/C. with occasional, gentle mixing.

3. BMMNC are washed twice in HHF and resuspended in 0.5 ml of HHFcontaining biotiixylated goat anti-mouse IgG (g-chain specific, SouthernBiotechnology Associates, Birmingham, UK) at a 1/50 dilution andincubated at 4/C for 45 minutes.

4. The BMMNC are washed three times in MACS buffer (Ca2+- and Mn2+-freePBS supplemented with 1% BSA in PBS, 5 mM EDTA and 0.01% sodium azide)and resuspended in 450 J. of MACS buffer to which 50 _l of streptavidinmicrobeads (Miltenyi Biotec; Bergisch Gladbaeh, Germany) are added (10 _of microbeads/107 cells in 90 J MACS buffer). The mixture is incubatedat 4/C for 15 minutes.

5. To monitor the purification process (optional), Strepavidin-PEconjugate ( 1/50) (Caltag Laboratories, San Francisco, Calif.) is addeddirectly to the cell suspension for an additional 5 minutes.

6. After 1 wash in ice-cold MACS buffer, a small aliquot of cells(approx. 200K) is removed for flow cytometric analysis (pre sample). Theremaining cells are then be placed onto the mini MACS column (columncapacity of 108 cells, Miltenyi Biotec, MS column). The TNAP− ceDs(negative fraction) are not retained within the column and pass through,under gravity into the effluent, whilst the TNAP+ cells remain, attachedto the magnetised matrix.

7. Wash the column 3 times with 0.5 ml MACs buffer to remove anynon-specifically bound TNAP− cells.

8. The TNAP+ cells are recovered by flushing the column with MACS bufferafter withdrawing the column from the magnetic field. Small samples fromeach of the pre, negative and positive fractions are removed, fixed inFACS Fix (1% (v/v) formalin, 0.1 M D-glucose, 0.02% sodium azide in PBS)and subsequently analysed by flow cytometry in order to assess purityand recovery.

Establishment and Ex Vivo Culture of STRO-3 mAb Selected Cells

1. The TNAP+ enriched populations (5×104 per cm2) are cultured in tissueculture flasks or plates containing, alpha-Modification of Eagles Medium(α-MEM) supplemented with 20% foetal bovine serum, 100 mML-ascorbate-2-phosphate, 2 mM L-glutamine, 50 U/ml penicillin and somg/ml streptomycin (CSL) at 37/C in 4% CO2 for two weeks.

2. Primary cell populations should be passaged when the cultures achieve80-90% confluency. Adherent cultures should be washed 1× with serum freeHBSS and the cells liberated by enzymatic digestion with 2 ml of 0.5%Trypsin/EDTA solution (IRE) per T75 flask for 5-10 minutes at 37° C.Cell suspensions are pooled and re-seeded at 0.5-1.0×104 per cm2 inα-MEM growth medium supplemented with 10% FBS.

3. Routinely, single cell suspensions of culture expanded cells areprepared by trypsin/EDTA digest as described above. The cells are thendiluted and washed in cold HFF. Following centrifugation, the cellpellet is resuspended at a concentration of 5×10⁶ cells per ml in FBSand maintained on ice. An equal volume of freeze mix (20% DMSO in coldFBS) is then added gradually while gently mixing the cells to give afinal concentration of 2.5×106 cells per ml in 10% DMSO/FBS. One mlaliquots are then distributed into 1.8 ml ciyovials (NUNC) on ice, i.e.1 ml per tube, then frozen at a rate of −loC per minute using a ratecontrol freezer. The frozen vials are then transferred to liquidnitrogen for long-term storage. Recovery of the frozen stocks isachieved by rapid thawing the cells in a 37 bC water bath. The cells arethen resuspended in cold HFF and spun at 280×g for 10 minutes. To assessviability of the cells, prepare a 1:5 dilution in 0.4% trypan blue/PBS,and the number of cells determined using, a haemocytometer. Typicallythis procedure gives viabilities between 80-90%.

Quality Control of Cells Preparations

1. BMSSC cultures are prepared by trypsin/EDTA digest then resuspendedin blocking buffer for 30 minutes.

2. Final cell qualification includes: negative gram stain, negativebacterial and fungal culture at 14 days, negative endotoxin testing, and70% viability by trypan-blue dye exclusion.

3. Cells are characterised by immunophenotype STRO-1+, TNAP+, CD146+,CD44+, CD3−, CD14−, colony forming assays (Colony FormingUnits-Fibroblasts (CFU-F), and induced osteoblast differentiation).

Autologous culture-expanded cells are then couriered, on ice, to theRoyal Melbourne Hospital, Melbourne, Australia, for implantation inpatients in need of bone regeneration, and to the John Hunter Hospitalin Newcastle, Australia, for intrarayocardial implantation in patientsin need of cardiac functional recovery and/or new blood vesselformation.

Percutaneous NOGA-Guided Bone Marrow Cell Implantation Procedure

The NOGA (Biosense) left ventricular electromechanical mapping systemutilises magnetic technology to navigate an 8 fr endocardial-mappingcatheter, which is introduced percutaneously via the right femoralartery and advanced into the left ventricle. The catheter is thendragged along the endocardial surface, acquiring local R wave electricalpotentials. The electrical signals are gated to surface ECG electrodes,thereby providing information on regional wall motion (local linearshortening scores). Areas of ischaemic but viable myocardium will bedetected by NOGA as a region of reduced local linear shortening butpreserved R wave potential. The pre-defined NOGA parameters of normalmyocardium is areas of electrical activity >5 mV and local linearshortening >12%. Infarcted myocardium will have areas of electricalactivity <5 mV and local linear shortening <4%. Ischaemic but viablemyocardium will have an electrical potential of 5 mV or greater andlocal linear shortening scores of 4-12%. The NOGA has been extensivelyvalidated as a tool for assessing myocardial viability on line in theCardiac Catheteiisation Laboratory. The Biosense Myo-Star TM injectioncatheter is similar to the mapping catheter as it has a magnetic sensorat its tip, which makes it locatable in space. It has a retractableneedle, which can be used to accurately inject the bone marrow cellsinto the target region. This system enables accurate and safe injectionof the bone marrow cells into the endocardium as it makes contact withthe endocardial surface.

Results and Discussion

Implantation Of STRO3-Selected and Culture-Expanded TNAP-Enriched Cellsin a Patient with a Non-Union Fracture of the Femur

A 19-year-old male presented to Hie Royal Melbourne Hospital with afracture of the femoral shaft which had occurred 9 months earlier due toa motorcycle accident and had failed to heal despite surgicalimplantation of rods and screws. A persistent, non-healing 5 cm defectpersisted.

Following informed consent, the patient underwent a bone marrowaspirate, with STRO-3 mAb selection of the bone marrow mononuclearcells, and culture-expansion of these cells, as above. Afterapproximately six weeks of culture, 200-225 million cells were harvestedand prepared for infusion surgically.

At infusion, cells were resuspended into sterile, ealine/plasmalyte to a5-10 ml volume and mixed with the Artificial Synthetic Bone Matrix(HA/TCP) containing bovine collagen (Mastergraft™ Matrix).

The procedure was uneventful, with no adverse events, and the defect wasfully closed.

Implantation of STRO3-Selected and Culture-Expanded TNAP-Enriched Cellsin Two Patients with Multiple Coronary Artery Vessel Occlusions andRefractory Chest Pain

Two males age ranges 40-65 years presented to the John Hunter Hospitalwith persistent chest pain on exertion and multivessel coronary arteryocclusions, not amenable to medical or surgical treatment.

Following informed consent, the patients underwent a bone marrowaspirate, with STRO-3 mAb selection of the bone marrow mononuclearcells, and culture-expansion of these cells, as above. Afterapproximately six weeks of culture, 100-120 million cells were harvestedfor each patient and prepared for mtramyocardial infusion by NOGA(Biosense) cardiac catheter.

For each patient, NOGA catheter-guided intramyocardial injection of thecultured cells was performed. At each target region, 10-12 injection of0.2 m containing cells was performed.

Echocardiograms were performed during and immediately after theprocedure in order to exclude perforation of the ventricular wall andensuing pericardial tamponade. After the procedure, patients wereobserved for 24 hours in the Coronary Care Unit. Electrocardiograms andcardiac enzyme levels were tested every 8 hours during Coronary CareUnit observation. Echocardiograms were obtained in the first 24 hrsafter implantation procedure.

No adverse events were observed either acutely or in the post-operativeperiod. The patients have each been followed for two months. During thisperiod, each patient has indicated reduced frequency and severity inepisodes of chest pain and reported increased exertional tolerance.

These data suggest that implantation of the expanded cells has resultedin increased vascular blood flow to the damaged and “at risk” areas ofmyocardium supplied by the occluded coronary vessels.

Example 14 Increased Cell Survival when Delivered with Fibrin GlueMaterials and Methods Preparation of Fibrin Glue

Fibrin Glue (Tisseal VH, Baxter) was prepared according to manufacturerspecification. Briefly, the vials containing the freeze-dried SealerProtein Concentrate, the Fibrinolysis Inhibitor Solution, and thethrombin were heated in a waterbath to 37° C. The Fibrinolysis InhibitorSolution was transferred into the vial containing the freeze-driedSealer Protein Concentrate using the sterile reconstitution componentsprovided with the DUPLOJECT Preparation and Application System. The vialwas allowed to stand at 37° C. for one minute then swirled briefly andvigorously with a circular motion (avoiding excessive frothing) andplaced into a water-bath for another minutes. The calcium chloridesolution was transferred to the thrombin solution once warmed.

Resuspension and Injection of Cells

Five million culture expanded immunoselected human MPCs in PBS weretransferred to the diluted thrombin solution. The thrombin/cellsolutions and reconstituted Sealer Protein solutions were then loadedinto a modified DUPLOJECT Application system loaded with two needleless,Ice insulin syringes (Beckton Dickinson) and a 27G ⅝″ needle (BecktonDickinson).

Forty-eight hours after LAD ligation and infarction of nude rats, theleft thoracotomy incision was reopened and adhesions were carefullylysed. The infarct zone was identified and 0.3 cc total volume(containing I million celts in 1:5 diluted fibrin glue) was injected inthree, equal divided doses into the peri-infaret region. The incisionwas closed in layers and the animal recovered. Following a furtherforty-eight hours animals were sacrificed, cardiac tissue obtained andtotal DNA was extracted by standard methods.

PCR of human β-globulin gene was performed on rat extracted cardiactissue DNA to estimate human cell survival from standard curves.

Results and Discussion

FIG. 24 shows that culture expanded MPCs of the invention demonstrateincreased survival in tissues in vivo when delivered in fribrin glue.

It will be appreciated by persons skilled in the art that numerousvariation and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments ate,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed above are incorporated herein in theirentirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

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1. Use of TNAP as a marker for the identification and/or enrichment ofadult multipotential cells.
 2. A method of enriching for adultmultipotential cells, the method comprising preparing a cell sample froma tissue source and enriching for adult multipotential cells thatexpress the TNAP marker.
 3. A method according to claim 2 whichcomprises: contacting the cell sample with a TNAP binding agent underconditions that allows binding of TNAP to the TNAP binding agent; andseparating cells bound to the TNAP binding agent. 4-5. (canceled)
 6. Amethod according to claim 3 wherein the TNAP binding agent bindsspecifically to the BAP isoform of TNAP.
 7. (canceled)
 8. A methodaccording to claim 6 wherein the anti-TNAP antibody is an antibody thatbinds to the same epitope as the STRO-3 antibody produced by thehybridoma cell line deposited with ATCC on 19 Dec. 2005 under theprovisions of the Budapest Treaty under deposit accession numberPTA-7282.
 9. (canceled)
 10. A method according to claim 8 wherein theanti-TNAP antibody is the STRO-3 antibody produced by the hybridoma cellline deposited with ATCC on 19 Dec. 2005 under the provisions of theBudapest Treaty under deposit accession number PTA-7282. 11-16.(canceled)
 17. A method according to claim 2 for identifying thepresence of an adult multipotential cell in a cell sample, the methodcomprising identifying cells in the sample that express the TNAP marker.18. An enriched population of adult multipotential cells obtained by amethod according to claim
 1. 19. An enriched population of TNAP+ adultmultipotential cells.
 20. An expanded cell population obtained byculturing an enriched population of adult multipotential cells accordingto claim
 18. 21. (canceled)
 22. A method of generating a tissue specificcommitted cell population, the method comprising culturing a populationof adult multipotential cells according to claim 18 in the presence ofone or more stimulatory factors; and subjecting said cultured populationto conditions biasing differentiation of the adult multipotential cellsto a specific tissue type. 23-24. (canceled)
 25. A compositioncomprising a population of enriched adult multipotential cells accordingto claim
 18. 26-28. (canceled)
 29. A method for generating or repairingtissue in a subject, the method comprising administering to the subjectan enriched or expanded cell population according to claim
 18. 30. Amethod for generating or repairing tissue in a subject, the methodcomprising administering to the subject a composition according to claim25.
 31. A method according to claim 29 wherein the tissue is bone,cardiac, or cartilage tissue. 32-37. (canceled)
 38. An isolated cellwhich has been obtained by a method according to claim 1, or a progenycell thereof, wherein the cell is genetically modified. 39-45.(canceled)